System for cell-based screening

ABSTRACT

The present invention provides systems, methods, and screens to measure receptor internalization in a single step with appropriate automation and throughput. This approach involves luminescent labeling of the receptor of interest and the automated measurement of receptor internalization to a perinuclear location.

CROSS REFERENCE

This application is a continuation-in-part of U.S. Ser. No. 09/031,271filed Feb. 27, 1998, now abandoned, which is a continuation in part ofU.S. Ser. No. 08/810,983 filed Feb. 27, 1997, now U.S. Pat. No.5,989,835. This application also claims priority to U.S. Applicationsfor patent Ser. No. 60/092,671, filed on Jul. 13, 1998.

FIELD OF THE INVENTION

This invention is in the field of fluorescence-based cell and molecularbiochemical assays for drug discovery.

BACKGROUND OF THE INVENTION

Drug discovery, as currently practiced in the art, is a long, multiplestep process involving identification of specific disease targets,development of an assay based on a specific target, validation of theassay, optimization and automation of the assay to produce a screen,high throughput screening of compound libraries using the assay toidentify “hits”, hit validation and hit compound optimization. Theoutput of this process is a lead compound that goes into pre-clinicaland, if validated, eventually into clinical trials. In this process, thescreening phase is distinct from the assay development phases, andinvolves testing compound efficacy in living biological systems.

Historically, drug discovery is a slow and costly process, spanningnumerous years and consuming hundreds of millions of dollars per drugcreated. Developments in the areas of genomics and high throughputscreening have resulted in increased capacity and efficiency in theareas of target identification and volume of compounds screened.Significant advances in automated DNA sequencing, PCR application,positional cloning, hybridization arrays, and bioinformatics havegreatly increased the number of genes (and gene fragments) encodingpotential drug screening targets. However, the basic scheme for drugscreening remains the same.

Validation of genomic targets as points for therapeutic interventionusing the existing methods and protocols has become a bottleneck in thedrug discovery process due to the slow, manual methods employed, such asin vivo functional models, functional analysis of recombinant proteins,and stable cell line expression of candidate genes. Primary DNA sequencedata acquired through automated sequencing does not permitidentification of gene function, but can provide information aboutcommon “motifs” and specific gene homology when compared to knownsequence databases. Genomic methods such as subtraction hybridizationand RADE (rapid amplification of differential expression) can be used toidentify genes that are up or down regulated in a disease state model.However, identification and validation still proceed down the samepathway. Some proteomic methods use protein identification (globalexpression arrays, 2D electrophoresis, combinatorial libraries) incombination with reverse genetics to identify candidate genes ofinterest. Such putative “disease associated sequences” or DAS isolatedas intact cDNA are a great advantage to these methods, but they areidentified by the hundreds without providing any information regardingtype, activity, and distribution of the encoded protein. Choosing asubset of DAS as drug screening targets is “random”, and thus extremelyinefficient, without functional data to provide a mechanistic link withdisease. It is necessary, therefore, to provide new technologies torapidly screen DAS to establish biological function, thereby improvingtarget validation and candidate optimization in drug discovery.

There are three major avenues for improving early drug discoveryproductivity. First, there is a need for tools that provide increasedinformation handling capability. Bioinformatics has blossomed with therapid development of DNA sequencing systems and the evolution of thegenomics database. Genomics is beginning to play a critical role in theidentification of potential new targets. Proteomics has becomeindispensable in relating structure and function of protein targets inorder to predict drug interactions. However, the next level ofbiological complexity is the cell. Therefore, there is a need toacquire, manage and search multi-dimensional information from cells.Secondly, there is a need for higher throughput tools. Automation is akey to improving productivity as has already been demonstrated in DNAsequencing and high throughput primary screening. The instant inventionprovides for automated systems that extract multiple parameterinformation from cells that meet the need for higher throughput tools.The instant invention also provides for miniaturizing the methods,thereby allowing increased throughput, while decreasing the volumes ofreagents and test compounds required in each assay.

Radioactivity has been the dominant read-out in early drug discoveryassays. However, the need for more information, higher throughput andminiaturization has caused a shift towards using fluorescence detection.Fluorescence-based reagents can yield more powerful, multiple parameterassays that are higher in throughput and information content and requirelower volumes of reagents and test compounds. Fluorescence is also saferand less expensive than radioactivity-based methods.

Screening of cells treated with dyes and fluorescent reagents is wellknown in the art. There is a considerable body of literature related togenetic engineering of cells to produce fluorescent proteins, such asmodified green fluorescent protein (GFP), as a reporter molecule. Someproperties of wild-type GFP are disclosed by Morise et al. (Biochemistry13 (1974), p. 2656-2662), and Ward et al. (Photochem. Photobiol. 31(1980), p. 611-615). The GFP of the jellyfish Aequorea Victoria has anexcitation maximum at 395 nm and an emission maximum at 510 nm, and doesnot require an exogenous factor for fluorescence activity. Uses for GFPdisclosed in the literature are widespread and include the study of geneexpression and protein localization (Chalfie et al., Science 263 (1994),p. 12501-12504)), as a tool for visualizing subcellular organelles(Rizzuto et al., Curr. Biology 5 (1995), p. 635-642)), visualization ofprotein transport along the secretory pathway (Kaether and Gerdes, FEBSLetters 369 (1995), p. 267-271)), expression in plant cells (Hu andCheng, FEBS Letters 369 (1995), p. 331-334)) and Drosophila embryos(Davis et al., Dev. Biology 170 (1995), p. 726-729)), and as a reportermolecule fused to another protein of interest (U.S. Pat. No. 5,491,084).Similarly, WO 96/23898 relates to methods of detecting biologicallyactive substances affecting intracellular processes by utilizing a GFPconstruct having a protein kinase activation site. This patent, and allother patents referenced in this application are incorporated byreference in their entirety

Numerous references are related to GFP proteins in biological systems.For example, WO 96/09598 describes a system for isolating cells ofinterest utilizing the expression of a GFP like protein. WO 96/27675describes the expression of GFP in plants. WO 95/21191 describesmodified GFP protein expressed in transformed organisms to detectmutagenesis. U.S. Pat. Nos. 5,401,629 and 5,436,128 describe assays andcompositions for detecting and evaluating the intracellular transductionof an extracellular signal using recombinant cells that express cellsurface receptors and contain reporter gene constructs that includetranscriptional regulatory elements that are responsive to the activityof cell surface receptors.

Performing a screen on many thousands of compounds requires parallelhandling and processing of many compounds and assay component reagents.Standard high throughput screens (“HTS”) use mixtures of compounds andbiological reagents along with some indicator compound loaded intoarrays of wells in standard microtiter plates with 96 or 384 wells. Thesignal measured from each well, either fluorescence emission, opticaldensity, or radioactivity, integrates the signal from all the materialin the well giving an overall population average of all the molecules inthe well.

Science Applications International Corporation (SAIC) 130 Fifth Avenue,Seattle, Wash. 98109) describes an imaging plate reader. This systemuses a CCD camera to image the whole area of a 96 well plate. The imageis analyzed to calculate the total fluorescence per well for all thematerial in the well.

Molecular Devices, Inc. (Sunnyvale, Calif.) describes a system (FLIPR)which uses low angle laser scanning illumination and a mask toselectively excite fluorescence within approximately 200 microns of thebottoms of the wells in standard 96 well plates in order to reducebackground when imaging cell monolayers. This system uses a CCD camerato image the whole area of the plate bottom. Although this systemmeasures signals originating from a cell monolayer at the bottom of thewell, the signal measured is averaged over the area of the well and istherefore still considered a measurement of the average response of apopulation of cells. The image is analyzed to calculate the totalfluorescence per well for cell-based assays. Fluid delivery devices havealso been incorporated into cell based screening systems, such as theFLIPR system, in order to initiate a response, which is then observed asa whole well population average response using a macro-imaging system.

In contrast to high throughput screens, various high-content screens(“HCS”) have been developed to address the need for more detailedinformation about the temporal-spatial dynamics of cell constituents andprocesses. High-content screens automate the extraction of multicolorfluorescence information derived from specific fluorescence-basedreagents incorporated into cells (Giuliano and Taylor (1995), Curr. Op.Cell Biol. 7:4; Giuliano et al. (1995) Ann. Rev. Biophys. Biomol.Struct. 24:405). Cells are analyzed using an optical system that canmeasure spatial, as well as temporal dynamics. (Farkas et al. (1993)Ann. Rev. Physiol. 55:785; Giuliano et al. (1990) In Optical Microscopyfor Biology. B. Herman and K. Jacobson (eds.), pp. 543-557. Wiley-Liss,New York; Hahn et al (1992) Nature 359:736; Waggoner et al. (1996) Hum.Pathol. 27:494). The concept is to treat each cell as a “well” that hasspatial and temporal information on the activities of the labeledconstituents.

The types of biochemical and molecular information now accessiblethrough fluorescence-based reagents applied to cells include ionconcentrations, membrane potential, specific translocations, enzymeactivities, gene expression, as well as the presence, amounts andpatterns of metabolites, proteins, lipids, carbohydrates, and nucleicacid sequences (DeBiasio et al., (1996) Mol. Biol. Cell. 7:1259;Giuliano et al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405; Heimand Tsien, (1996) Curr. Biol. 6:178).

High-content screens can be performed on either fixed cells, usingfluorescently labeled antibodies, biological ligands, and/or nucleicacid hybridization probes, or live cells using multicolor fluorescentindicators and “biosensors.” The choice of fixed or live cell screensdepends on the specific cell-based assay required.

Fixed cell assays are the simplest, since an array of initially livingcells in a microtiter plate format can be treated with various compoundsand doses being tested, then the cells can be fixed, labeled withspecific reagents, and measured. No environmental control of the cellsis required after fixation. Spatial information is acquired, but only atone time point. The availability of thousands of antibodies, ligands andnucleic acid hybridization probes that can be applied to cells makesthis an attractive approach for many types of cell-based screens. Thefixation and labeling steps can be automated, allowing efficientprocessing of assays.

Live cell assays are more sophisticated and powerful, since an array ofliving cells containing the desired reagents can be screened over time,as well as space. Environmental control of the cells (temperature,humidity, and carbon dioxide) is required during measurement, since thephysiological health of the cells must be maintained for multiplefluorescence measurements over time. There is a growing list offluorescent physiological indicators and “biosensors” that can reportchanges in biochemical and molecular activities within cells (Giulianoet al., (1995) Ann. Rev. Biophys. Biomol. Struct. 24:405; Hahn et al.,(1993) In Fluorescent and Luminescent Probes for Biological Activity. W.T. Mason, (ed.), pp. 349-359, Academic Press, San Diego).

The availability and use of fluorescence-based reagents has helped toadvance the development of both fixed and live cell high-contentscreens. Advances in instrumentation to automatically extractmulticolor, high-content information has recently made it possible todevelop HCS into an automated tool. An article by Taylor, et al.(American Scientist 80 (1992), p. 322-335) describes many of thesemethods and their applications. For example, Proffitt et. al. (Cytometry24: 204-213 (1996)) describe a semi-automated fluorescence digitalimaging system for quantifying relative cell numbers in situ in avariety of tissue culture plate formats, especially 96-well microtiterplates. The system consists of an epifluorescence inverted microscopewith a motorized stage, video camera, image intensifier, and amicrocomputer with a PC-Vision digitizer. Turbo Pascal software controlsthe stage and scans the plate taking multiple images per well. Thesoftware calculates total fluorescence per well, provides for dailycalibration, and configures easily for a variety of tissue culture plateformats. Thresholding of digital images and reagents which fluoresceonly when taken up by living cells are used to reduce backgroundfluorescence without removing excess fluorescent reagent.

Scanning confocal microscope imaging (Go et al., (1997) AnalyticalBiochemistry 247:210-215; Goldman et al., (1995) Experimental CellResearch 221:311-319) and multiphoton microscope imaging (Denk et al.,(1990) Science 248:73; Gratton et al., (1994) Proc. of the MicroscopicalSociety of America, pp. 154-155) are also well established methods foracquiring high resolution images of microscopic samples. The principleadvantage of these optical systems is the very shallow depth of focus,which allows features of limited axial extent to be resolved against thebackground. For example, it is possible to resolve internal cytoplasmicfeatures of adherent cells from the features on the cell surface.Because scanning multiphoton imaging requires very short duration pulsedlaser systems to achieve the high photon flux required, fluorescencelifetimes can also be measured in these systems (Lakowicz et al., (1992)Anal. Biochem. 202:316-330; Gerrittsen et al. (1997), J. of Fluorescence7:11-15)), providing additional capability for different detectionmodes. Small, reliable and relatively inexpensive laser systems, such aslaser diode pumped lasers, are now available to allow multiphotonconfocal microscopy to be applied in a fairly routine fashion.

A combination of the biological heterogeneity of cells in populations(Bright, et al., (1989). J. Cell. Physiol. 141:410; Giuliano, (1996)Cell Motil. Cytoskel. 35:237)) as well as the high spatial and temporalfrequency of chemical and molecular information present within cells,makes it impossible to extract high-content information from populationsof cells using existing whole microtiter plate readers. No existinghigh-content screening platform has been designed for multicolor,fluorescence-based screens using cells that are analyzed individually.Similarly, no method is currently available that combines automatedfluid delivery to arrays of cells for the purpose of systematicallyscreening compounds for the ability to induce a cellular response thatis identified by HCS analysis, especially from cells grown in microtiterplates. Furthermore, no method exists in the art combining highthroughput well-by-well measurements to identify “hits” in one assayfollowed by a second high content cell-by-cell measurement on the sameplate of only those wells identified as hits.

The instant invention provides systems, methods, and screens thatcombine high throughput screening (HTS) and high content screening (HCS)that significantly improve target validation and candidate optimizationby combining many cell screening formats with fluorescence-basedmolecular reagents and computer-based feature extraction, data analysis,and automation, resulting in increased quantity and speed of datacollection, shortened cycle times, and, ultimately, faster evaluation ofpromising drug candidates. The instant invention also provides forminiaturizing the methods, thereby allowing increased throughput, whiledecreasing the volumes of reagents and test compounds required in eachassay.

SUMMARY OF THE INVENTION

The present invention provides fully automated methods for measuring andanalyzing cell surface receptor protein internalization during imageacquisition. This approach involves fluorescent labeling of the receptorof interest and the automated measurement of receptor internalization instimulated cells.

In one aspect of the present invention, methods, computer readablestorage medium, and kits are provided for identifying compounds thatinduce or inhibit internalization of cell surface receptor proteins,comprising treating cells that possess a luminescently-tagged cellsurface receptor protein with a test compound, obtaining luminescentsignals from the cells, converting the luminescent signals into digitaldata, and utilizing the digital data to determine whether the testcompound has induced internalization of the luminescently labeled cellsurface receptor protein into the cell.

Various preferred embodiments are provided, that allow for improvedspatial resolution and quantitation of the stimulatory or inhibitoryeffect of the test compound on receptor internalization. In one suchembodiment, the extracellular and intracellular domains of a membranebound receptor protein are each labeled with a distinct luminescentmarker, to permit measuring the relative extracellular availability ofexternal and internal domains of membrane receptors.

In another aspect of the invention, combined high throughput and highcontent methods and associated computer readable storage medium areprovided for identifying compounds that induce or inhibitinternalization of cell surface receptor proteins. In this aspect, cellsare treated with a ligand for the receptor protein of interest, whichproduces a detectable signal upon stimulation of the receptor protein.The cells are then treated with the test compound, and then scanned in ahigh throughput mode to identify those cells that exhibit theligand-induced detectable signal. Subsequently, only those cells thatexhibited the detectable signal are scanned in a high content mode, todetermine whether the test compound has induced internalization of theluminescently labeled cell surface receptor protein into the cell.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a diagram of the components of the cell-based scanningsystem.

FIG. 2 shows a schematic of the microscope subassembly.

FIG. 3 shows the camera subassembly.

FIG. 4 illustrates cell scanning system process.

FIG. 5 illustrates a user interface showing major functions to guide theuser.

FIG. 6 is a block diagram of the two platform architecture of the DualMode System for Cell Based Screening in which one platform uses atelescope lens to read all wells of a microtiter plate and a secondplatform that uses a higher magnification lens to read individual cellsin a well.

FIG. 7 is a detail of an optical system for a single platformarchitecture of the Dual Mode System for Cell Based Screening that usesa moveable ‘telescope’ lens to read all wells of a microtiter plate anda moveable higher magnification lens to read individual cells in a well.

FIG. 8 is an illustration of the fluid delivery system for acquiringkinetic data on the Cell Based Screening System.

FIG. 9 is a flow chart of processing step for the cell-based scanningsystem.

FIGS. 10A-J illustrates the strategy of the Nuclear Translocation Assay.

FIG. 11 is a flow chart defining the processing steps in the Dual ModeSystem for Cell Based Screening combining high throughput and highcontent screening of microtiter plates.

FIG. 12 is a flow chart defining the processing steps in the HighThroughput mode of the System for Cell Based Screening.

FIG. 13 is a flow chart defining the processing steps in the HighContent mode of the System for Cell Based Screening.

FIG. 14 is a flow chart defining the processing steps required foracquiring kinetic data in the High Content mode of the System for CellBased Screening.

FIG. 15 is a flow chart defining the processing steps performed within awell during the acquisition of kinetic data.

FIG. 16 is an example of data from a known inhibitor of translocation.

FIG. 17 is an example of data from a known stimulator of translocation.

FIG. 18 illustrates data presentation on a graphical display.

FIG. 19 is an illustration of the data from the High Throughput mode ofthe System for Cell Based Screening, an example of the data passed tothe High Content mode, the data acquired in the high content mode, andthe results of the analysis of that data.

FIG. 20 shows the measurement of a drug-induced cytoplasm to nucleartranslocation.

FIG. 21 illustrates a graphical user interface of the measurement shownin FIG. 20.

FIG. 22 illustrates a graphical user interface of the measurement shownin FIG. 20.

FIG. 23 is a graph representing the kinetic data obtained from themeasurements depicted in FIG. 20.

FIG. 24 details a high-content screen of drug-induced apoptosis.

FIG. 25 is a graphical representation of data from validation runs ofthe PTHR internalization screen.

FIG. 26 is a flow chart for signal processing.

FIG. 27 is a flow chart for an autofocusing procedure to be used insignal processing.

FIG. 28 is a flow chart for object processing procedure to be used insignal processing.

FIG. 29 shows a representative display of a PC screen showing receptorinternalization data displaying the spot count of individual wells.

FIG. 30 shows a representative display of a PC screen showing receptorinternalization data displayed on a field by field basis.

DETAILED DESCRIPTION OF THE INVENTION

All cited patents, patent applications and other references are herebyincorporated by reference in their entirety. As used herein, thefollowing terms have the specified meaning:

Markers of cellular domains. Luminescent probes that have high affinityfor specific cellular constituents including specific organelles ormolecules. These probes can either be small luminescent molecules orfluorescently tagged macromolecules used as “labeling reagents”,“environmental indicators”, or “biosensors.”

Labeling reagents. Labeling reagents include, but are not limited to,luminescently labeled macromolecules including fluorescent proteinanalogs and biosensors, luminescent macromolecular chimeras includingthose formed with the green fluorescent protein and mutants thereof,luminescently labeled primary or secondary antibodies that react withcellular antigens involved in a physiological response, luminescentstains, dyes, and other small molecules.

Markers of cellular translocations. Luminescently tagged macromoleculesor organelles that move from one cell domain to another during somecellular process or physiological response. Translocation markers caneither simply report location relative to the markers of cellulardomains or they can also be “biosensors” that report some biochemical ormolecular activity as well.

Biosensors. Macromolecules consisting of a biological functional domainand a luminescent probe or probes that report the environmental changesthat occur either internally or on their surface. A class ofluminescently labeled macromolecules designed to sense and report thesechanges have been termed “fluorescent-protein biosensors”. The proteincomponent of the biosensor provides a highly evolved molecularrecognition moiety. A fluorescent molecule attached to the proteincomponent in the proximity of an active site transduces environmentalchanges into fluorescence signals that are detected using a system withan appropriate temporal and spatial resolution such as the cell scanningsystem of the present invention. Because the modulation of nativeprotein activity within the living cell is reversible, and becausefluorescent-protein biosensors can be designed to sense reversiblechanges in protein activity, these biosensors are essentially reusable.

Disease associated sequences (“DAS”). This term refers to nucleic acidsequences identified by standard techniques, such as primary DNAsequence data, genomic methods such as subtraction hybridization andRADE, and proteomic methods in combination with reverse genetics, asbeing of drug candidate compounds. The term does not mean that thesequence is only associated with a disease state.

High content screening (HCS) can be used to measure the effects of drugson complex molecular events such as signal transduction pathways, aswell as cell functions including, but not limited to, apoptosis, celldivision, cell adhesion, locomotion, exocytosis, and cell-cellcommunication. Multicolor fluorescence permits multiple targets and cellprocesses to be assayed in a single screen. Cross-correlation ofcellular responses will yield a wealth of information required fortarget validation and lead optimization.

In one aspect of the present invention, a cell screening system isprovided comprising a high magnification fluorescence optical systemhaving a microscope objective, an XY stage adapted for holding a platewith an array of locations for holding cells and having a means formoving the plate to align the locations with the microscope objectiveand a means for moving the plate in the direction to effect focusing; adigital camera; a light source having optical means for directingexcitation light to cells in the array of locations and a means fordirecting fluorescent light emitted from the cells to the digitalcamera; and a computer means for receiving and processing digital datafrom the digital camera wherein the computer means includes: a digitalframe grabber for receiving the images from the camera, a display foruser interaction and display of assay results, digital storage media fordata storage and archiving, and means for control, acquisition,processing and display of results.

FIG. 1 is a schematic diagram of a preferred embodiment of the cellscanning system. An inverted fluorescence microscope is used 1, such asa Zeiss Axiovert inverted fluorescence microscope which uses standardobjectives with magnification of 1-100× to the camera, and a white lightsource (e.g. 100W mercury-arc lamp or 75W xenon lamp) with power supply2. There is an XY stage 3 to move the plate 4 in the XY direction overthe microscope objective. A Z-axis focus drive 5 moves the objective inthe Z direction for focusing. A joystick 6 provides for manual movementof the stage in the XYZ direction. A high resolution digital camera 7acquires images from each well or location on the plate. There is acamera power supply 8, an automation controller 9 and a centralprocessing unit 10. The PC 11 provides a display 12 and has associatedsoftware. The printer 13 provides for printing of a hard copy record.

FIG. 2 is a schematic of one embodiment of the microscope assembly 1 ofthe invention, showing in more detail the XY stage 3, Z-axis focus drive5, joystick 6, light source 2, and automation controller 9. Cables tothe computer 15 and microscope 16, respectively, are provided. Inaddition, FIG. 2 shows a 96 well microtiter plate 17 which is moved onthe XY stage 3 in the XY direction. Light from the light source 2 passesthrough the PC controlled shutter 18 to a motorized filter wheel 19 withexcitation filters 20. The light passes into filter cube 25 which has adichroic mirror 26 and an emission filter 27. Excitation light reflectsoff the dichroic mirror to the wells in the microtiter plate 17 andfluorescent light 28 passes through the dichroic mirror 26 and theemission filter 27 and to the digital camera 7.

FIG. 3 shows a schematic drawing of a preferred camera assembly. Thedigital camera 7, which contains an automatic shutter for exposurecontrol and a power supply 31, receives fluorescent light 28 from themicroscope assembly. A digital cable 30 transports digital signals tothe computer.

The standard optical configurations described above use microscopeoptics to directly produce an enlarged image of the specimen on thecamera sensor in order to capture a high resolution image of thespecimen. This optical system is commonly referred to as ‘wide field’microscopy. Those skilled in the art of microscopy will recognize that ahigh resolution image of the specimen can be created by a variety ofother optical systems, including, but not limited to, standard scanningconfocal detection of a focused point or line of illumination scannedover the specimen (Go et al. 1997, supra), and multi-photon scanningconfocal microscopy (Denk et al., 1990, supra), both of which can formimages on a CCD detector or by synchronous digitization of the analogoutput of a photomultiplier tube.

In screening applications, it is often necessary to use a particularcell line, or primary cell culture, to take advantage of particularfeatures of those cells. Those skilled in the art of cell culture willrecognize that some cell lines are contact inhibited, meaning that theywill stop growing when they become surrounded by other cells, whileother cell lines will continue to grow under those conditions and thecells will literally pile up, forming many layers. An example of such acell line is the HEK 293 (ATCC CRL-1573) line. An optical system thatcan acquire images of single cell layers in multilayer preparations isrequired for use with cell lines that tend to form layers. The largedepth of field of wide field microscopes produces an image that is aprojection through the many layers of cells, making analysis ofsubcellular spatial distributions extremely difficult in layer-formingcells. Alternatively, the very shallow depth of field that can beachieved on a confocal microscope, (about one micron), allowsdiscrimination of a single cell layer at high resolution, simplifyingthe determination of the subcellular spatial distribution. Similarly,confocal imaging is preferable when detection modes such as fluorescencelifetime imaging are required.

The output of a standard confocal imaging attachment for a microscope isa digital image that can be converted to the same format as the imagesproduced by the other cell screening system embodiments described above,and can therefore be processed in exactly the same way as those images.The overall control, acquisition and analysis in this embodiment isessentially the same. The optical configuration of the confocalmicroscope system, is essentially the same as that described above,except for the illuminator and detectors. Illumination and detectionsystems required for confocal microscopy have been designed asaccessories to be attached to standard microscope optical systems suchas that of the present invention (Zeiss, Germany). These alternativeoptical systems therefore can be easily integrated into the system asdescribed above.

FIG. 4 illustrates an alternative embodiment of the invention in whichcell arrays are in microwells 40 on a microplate 41, described inco-pending U.S. Pat. No. 6,103,479, incorporated by reference herein inits entirety. Typically the microplate is 20 mm by 30 mm as compared toa standard 96 well microtiter plate which is 86 mm by 129 mm. The higherdensity array of cells it on a microplate allows the microplate to beimaged at a low resolution of a few microns per pixel for highthroughput and particular locations on the microplate to be imaged at ahigher resolution of less than 0.5 microns per pixel. These tworesolution modes help to improve the overall throughput of the system.

The microplate chamber 42 serves as a microfluidic delivery system forthe addition of compounds to cells. The microplate 41 in the microplatechamber 42 is placed in an XY microplate reader 43. Digital data isprocessed as described above. The small size of this microplate systemincreases throughput, minimizes reagent volume and allows control of thedistribution and placement of cells for fast and precise cell-basedanalysis. Processed data can be displayed on a PC screen 11 and madepart of a bioinformatics data base 44. This data base not only permitsstorage and retrieval of data obtained through the methods of thisinvention, but also permits acquisition and storage of external datarelating to cells. FIG. 5 is a PC display which illustrates theoperation of the software.

In an alternative embodiment, a high throughput system (HTS) is directlycoupled with the HCS either on the same platform or on two separateplatforms connected electronically (e.g. via a local area network). Thisembodiment of the invention, referred to as a dual mode optical system,has the advantage of increasing the throughput of a HCS by coupling itwith a HTS and thereby requiring slower high resolution data acquisitionand analysis only on the small subset of wells that show a response inthe coupled HTS.

High throughput ‘whole plate’ reader systems are well known in the artand are commonly used as a component of an HTS system used to screenlarge numbers of compounds (Beggs (1997), J. of Biomolec. Screening2:71-78; Macaffrey et al., (1996) J. Biomolec. Screening 1:187-190).

In one embodiment of dual mode cell based screening, a two platformarchitecture in which high throughput acquisition occurs on one platformand high content acquisition occurs on a second platform is provided(FIG. 6). Processing occurs on each platform independently, with resultspassed over a network interface, or a single controller is used toprocess the data from both platforms.

As illustrated in FIG. 6, an exemplified two platform dual mode opticalsystem consists of two light optical instruments, a high throughputplatform 60 and a high content platform 65, which read fluorescentsignals emitted from cells cultured in microtiter plates or microwellarrays on a microplate, and communicate with each other via anelectronic connection 64. The high throughput platform 60 analyzes allthe wells in the whole plate either in parallel or rapid serial fashion.Those skilled in the art of screening will recognize that there are amany such commercially available high throughput reader systems thatcould be integrated into a dual mode cell based screening system(Topcount (Packard Instruments, Meriden, Conn.); Spectramax, Lumiskan(Molecular Devices, Sunnyvale, Calif.); Fluoroscan (Labsystems, Beverly,Mass.)). The high content platform 65, as described above, scans fromwell to well and acquires and analyzes high resolution image datacollected from individual cells within a well.

The HTS software, residing on the system's computer 62, controls thehigh throughput instrument, and results are displayed on the monitor 61.The HCS software, residing on it's computer system 67, controls the highcontent instrument hardware 65, optional devices (e.g. plate loader,environmental chamber, fluid dispenser), analyzes digital image datafrom the plate, displays results on the monitor 66 and manages datameasured in an integrated database. The two systems can also share asingle computer, in which case all data would be collected, processedand displayed on that computer, without the need for a local areanetwork to transfer the data. Microtiter plates are transferred from thehigh throughput system to the high content system 63 either manually orby a robotic plate transfer device, as is well known in the art (Beggs(1997), supra; Mcaffrey (1996), supra).

In a preferred embodiment, the dual mode optical system utilizes asingle platform system (FIG. 7). It consists of two separate opticalmodules, an HCS module 203 and an HTS module 209 that can beindependently or collectively moved so that only one at a time is usedto collect data from the microtiter plate 201. The microtiter plate 201is mounted in a motorized X,Y stage so it can be positioned for imagingin either HTS or HCS mode. After collecting and analyzing the HTS imagedata as described below, the HTS optical module 209 is moved out of theoptical path and the HCS optical module 203 is moved into place.

The optical module for HTS 209 consists of a projection lens 214,excitation wavelength filter 213 and dichroic mirror 210 which are usedto illuminate the whole bottom of the plate with a specific wavelengthband from a conventional microscope lamp system (not illustrated). Thefluorescence emission is collected through the dichroic mirror 210 andemission wavelength filter 211 by a lens 212 which forms an image on thecamera 216 with sensor 215.

The optical module for HCS 203 consists of a projection lens 208,excitation wavelength filter 207 and dichroic mirror 204 which are usedto illuminate the back aperture of the microscope objective 202, andthereby the field of that objective, from a standard microscopeillumination system (not shown). The fluorescence emission is collectedby the microscope objective 202, passes through the dichroic mirror 204and emission wavelength filter 205 and is focused by a tube lens 206which forms an image on the same camera 216 with sensor 215.

In an alternative embodiment of the present invention, the cellscreening system further comprises a fluid delivery device for use withthe live cell embodiment of the method of cell screening (see below).FIG. 8 exemplifies a fluid delivery device for use with the system ofthe invention. It consists of a bank of 12 syringe pumps 701 driven by asingle motor drive. Each syringe 702 is sized according to the volume tobe delivered to each well, typically between 1 and 100 μL. Each syringeis attached via flexible tubing 703 to a similar bank of connectorswhich accept standard pipette tips 705. The bank of pipette tips areattached to a drive system so they can be lowered and raised relative tothe microtiter plate 706 to deliver fluid to each well. The plate ismounted on an X,Y stage, allowing movement relative to the opticalsystem 707 for data collection purposes. This set-up allows one set ofpipette tips, or even a single pipette tip, to deliver reagent to allthe wells on the plate. The bank of syringe pumps can be used to deliverfluid to 12 wells simultaneously, or to fewer wells by removing some ofthe tips.

In another aspect, the present invention provides a method for analyzingcells comprising providing an array of locations which contain multiplecells wherein the cells contain one or more fluorescent reportermolecules; scanning multiple cells in each of the locations containingcells to obtain fluorescent signals from the fluorescent reportermolecule in the cells; converting the fluorescent signals into digitaldata; and utilizing the digital data to determine the distribution,environment or activity of the fluorescent reporter molecule within thecells.

Cell Arrays

Screening large numbers of compounds for activity with respect to aparticular biological function requires preparing arrays of cells forparallel handling of cells and reagents. Standard 96 well microtiterplates which are 86 mm by 129 mm, with 6 mm diameter wells on a 9 nmpitch, are used for compatibility with current automated loading androbotic handling systems. The microplate is typically 20 mm by 30 mm,with cell locations that are 100-200 microns in dimension on a pitch ofabout 500 microns. Methods for making microplates are described in U.S.Pat. No. 6,103,479, incorporated by reference herein in its entirety.Microplates may consist of coplanar layers of materials to which cellsadhere, patterned with materials to which cells will not adhere, oretched 3-dimensional surfaces of similarly pattered materials. For thepurpose of the following discussion, the terms ‘well’ and ‘microwell’refer to a location in an array of any construction to which cellsadhere and within which the cells are imaged. Microplates may alsoinclude fluid delivery channels in the spaces between the wells. Thesmaller format of a microplate increases the overall efficiency of thesystem by minimizing the quantities of the reagents, storage andhandling during preparation and the overall movement required for thescanning operation. In addition, the whole area of the microplate can beimaged more efficiently, allowing a second mode of operation for themicroplate reader as described later in this document.

Fluorescence Reporter Molecules

A major component of the new drug discovery paradigm is a continuallygrowing family of fluorescent and luminescent reagents that are used tomeasure the temporal and spatial distribution, content, and activity ofintracellular ions, metabolites, macromolecules, and organelles. Classesof these reagents include labeling reagents that measure thedistribution and amount of molecules in living and fixed cells,environmental indicators to report signal transduction events in timeand space, and fluorescent protein biosensors to measure targetmolecular activities within living cells. A multiparameter approach thatcombines several reagents in a single cell is a powerful new tool fordrug discovery.

The method of the present invention is based on the high affinity offluorescent or luminescent molecules for specific cellular components.The affinity for specific components is governed by physical forces suchas ionic interactions, covalent bonding (which includes chimeric fusionwith protein-based chromophores, fluorophores, and lumiphores), as wellas hydrophobic interactions, electrical potential, and, in some cases,simple entrapment within a cellular component. The luminescent probescan be small molecules, labeled macromolecules, or geneticallyengineered proteins, including, but not limited to green fluorescentprotein chimeras.

Those skilled in this art will recognize a wide variety of fluorescentreporter molecules that can be used in the present invention, including,but not limited to, fluorescently labeled biomolecules such as proteins,phospholipids and DNA hybridizing probes. Similarly, fluorescentreagents specifically synthesized with particular chemical properties ofbinding or association have been used as fluorescent reporter molecules(Barak et al., (1997), J. Biol. Chem. 272:27497-27500; Southwick et al.,(1990), Cytometry 11:418-430; Tsien (1989) in Methods in Cell Biology,Vol. 29 Taylor and Wang (eds.), pp. 127-156). Fluorescently labeledantibodies are particularly useful reporter molecules due to their highdegree of specificity for attaching to a single molecular target in amixture of molecules as complex as a cell or tissue.

The luminescent probes can be synthesized within the living cell or canbe transported into the cell via several non-mechanical modes includingdiffusion, facilitated or active transport, signal-sequence-mediatedtransport, and endocytotic or pinocytotic uptake. Mechanical bulkloading methods, which are well known in the art, can also be used toload luminescent probes into living cells (Barber et al. (1996),Neuroscience Letters 207:17-20; Bright et al. (1996), Cytometry24:226-233; McNeil (1989) in Methods in Cell Biology, Vol. 29, Taylorand Wang (eds.), pp. 153-173). These methods include electroporation andother mechanical methods such as scrape-loading, bead-loading,impact-loading, syringe-loading, hypertonic and hypotonic loading.Additionally, cells can be genetically engineered to express reportermolecules, such as GFP, coupled to a protein of interest as previouslydescribed (Chalfie and Prasher U.S. Pat. No. 5,491,084; Cubitt et al.(1995), Trends in Biochemical Science 20:448-455).

Once in the cell, the luminescent probes accumulate at their targetdomain as a result of specific and high affinity interactions with thetarget domain or other modes of molecular targeting such assignal-sequence-mediated transport. Fluorescently labeled reportermolecules are useful for determining the location, amount and chemicalenvironment of the reporter. For example, whether the reporter is in alipophilic membrane environment or in a more aqueous environment can bedetermined (Giuliano et al. (1995), Ann. Rev. of Biophysics andBiomolecular Structure 24:405-434; Giuliano and Taylor (1995), Methodsin Neuroscience 27:1-16). The pH environment of the reporter can bedetermined (Bright et al. (1989), J. Cell Biology 104:1019-1033;Giuliano et al. (1987), Anal. Biochem. 167:362-371; Thomas et al.(1979), Biochemistry 18:2210-2218). It can be determined whether areporter having a chelating group is bound to an ion, such as Ca++, ornot (Bright et al. (1989), In Methods in Cell Biology, Vol. 30, Taylorand Wang (eds.), pp. 157-192; Shimoura et al. (1988), J. of Biochemistry(Tokyo) 251:405-410; Tsien (1989) In Methods in Cell Biology, Vol. 30,Taylor and Wang (eds.), pp. 127-156).

Furthermore, certain cell types within an organism may containcomponents that can be specifically labeled that may not occur in othercell types. For example, epithelial cells often contain polarizedmembrane components. That is, these cells asymmetrically distributemacromolecules along their plasma membrane. Connective or supportingtissue cells often contain granules in which are trapped moleculesspecific to that cell type (e.g., heparin, histamine, serotonin, etc.).Most muscular tissue cells contain a sarcoplasmic reticulum, aspecialized organelle whose function is to regulate the concentration ofcalcium ions within the cell cytoplasm. Many nervous tissue cellscontain secretory granules and vesicles in which are trappedneurohormones or neurotransmitters. Therefore, fluorescent molecules canbe designed to label not only specific components within specific cells,but also specific cells within a population of mixed cell types.

Those skilled in the art will recognize a wide variety of ways tomeasure fluorescence. For example, some fluorescent reporter moleculesexhibit a change in excitation or emission spectra, some exhibitresonance energy transfer where one fluorescent reporter losesfluorescence, while a second gains in fluorescence, some exhibit a loss(quenching) or appearance of fluorescence, while some report rotationalmovements (Giuliano et al. (1995), Ann. Rev. of Biophysics and Biomol.Structure 24:405-434; Giuliano et al. (1995), Methods in Neuroscience27:1-16).

Scanning Cell Arrays

Referring to FIG. 9, a preferred embodiment is provided to analyze cellsthat comprises operator-directed parameters being selected based on theassay being conducted, data acquisition by the cell screening system onthe distribution of fluorescent signals within a sample, and interactivedata review and analysis. At the start of an automated scan the operatorenters information 100 that describes the sample, specifies the filtersettings and fluorescent channels to match the biological labels beingused and the information sought, and then adjusts the camera settings tomatch the sample brightness. For flexibility to handle a range ofsamples, the software allows selection of various parameter settingsused to identify nuclei and cytoplasm, and selection of differentfluorescent reagents, identification of cells of interest based onmorphology or brightness, and cell numbers to be analyzed per well.These parameters are stored in the system's database for easy retrievalfor each automated run. The system's interactive cell identificationmode simplifies the selection of morphological parameter limits such asthe range of size, shape, and intensity of cells to be analyzed. Theuser specifies which wells of the plate the system will scan and howmany fields or how many cells to analyze in each well. Depending on thesetup mode selected by the user at step 101, the system eitherautomatically pre-focuses the region of the plate to be scanned using anautofocus procedure to “find focus” of the plate 102 or the userinteractively pre-focuses 103 the scanning region by selecting three“tag” points which define the rectangular area to be scanned. Aleast-squares fit “focal plane model” is then calculated from these tagpoints to estimate the focus of each well during an automated scan. Thefocus of each well is estimated by interpolating from the focal planemodel during a scan.

During an automated scan, the software dynamically displays the scanstatus, including the number of cells analyzed, the current well beinganalyzed, images of each independent wavelength as they are acquired,and the result of the screen for each well as it is determined. Theplate 4 (FIG. 1) is scanned in a serpentine style as the softwareautomatically moves the motorized microscope XY stage 3 from swell towell and field to field within each well of a 96-well plate. Thoseskilled in the programming art will recognize how to adapt software forscanning of other microplate formats such as 24, 48, and 384 wellplates. The scan pattern of the entire plate as well as the scan patternof fields within each well are programmed. The system adjusts samplefocus with an autofocus procedure 104 (FIG. 9) through the Z axis focusdrive 5, controls filter selection via a motorized filter wheel 19, andacquires and analyzes images of up to four different colors (“channels”or “wavelengths”).

The autofocus procedure is called at a user selected frequency,typically for the first field in each well and then once every 4 to 5fields within each well. The autofocus procedure calculates the startingZ-axis point by interpolating from the pre-calculated plane focal model.Starting a programmable distance above or below this set point, theprocedure moves the mechanical Z-axis through a number of differentpositions, acquires an image at each position, and finds the maximum ofa calculated focus score that estimates the contrast of each image. TheZ position of the image with the maximum focus score determines the bestfocus for a particular field. Those skilled in the art will recognizethis as a variant of automatic focusing algorithms as described in Harmset al. in Cytometry 5 (1984), 236-243, Groen et al. in Cytometry 6(1985), 81-91, and Firestone et al. in Cytometry 12 (1991), 195-206.

For image acquisition, the camera's exposure time is separately adjustedfor each dye to ensure a high-quality image from each channel. Softwareprocedures can be called, at the user's option, to correct forregistration shifts between wavelengths by accounting for linear (X andY) shifts between wavelengths before making any further measurements.The electronic shutter 18 is controlled so that sample photo-bleachingis kept to a minimum. Background shading and uneven illumination can becorrected by the software using methods known in the art (Bright et al.(1987), J. Cell Biol. 104:1019-1033).

In one channel, images are acquired of a primary marker 105 (FIG. 9)(typically cell nuclei counterstained with DAPI or PI fluorescent dyes)which are segmented (“identified”) using an adaptive thresholdingprocedure. The adaptive thresholding procedure 106 is used todynamically select the threshold of an image for separating cells fromthe background. The staining of cells with fluorescent dyes can vary toan unknown degree across cells in a microtiter plate sample as well aswithin images of a field of cells within each well of a microtiterplate. This variation can occur as a result of sample preparation and/orthe dynamic nature of cells. A global threshold is calculated for thecomplete image to separate the cells from background and account forfield to field variation. These global adaptive techniques are variantsof those described in the art. (Kittler et al. in Computer Vision,Graphics, and Image Processing 30 (1985), 125-147, Ridler et al. in IEEETrans. Systems, Man, and Cybernetics (1978), 630-632.)

An alternative adaptive thresholding method utilizes local regionthresholding in contrast to global image thresholding. Image analysis oflocal regions leads to better overall segmentation since staining ofcell nuclei (as well as other labeled components) can vary across animage. Using this global/local procedure, a reduced resolution image(reduced in size by a factor of 2 to 4) is first globally segmented(using adaptive thresholding) to find regions of interest in the image.These regions then serve as guides to more fully analyze the sameregions at full resolution. A more localized threshold is thencalculated (again using adaptive thresholding) for each region ofinterest.

The output of the segmentation procedure is a binary image wherein theobjects are white and the background is black. This binary image, alsocalled a mask in the art, is used to determine if the field containsobjects 107. The mask is labeled with a blob labeling algorithm wherebyeach object (or blob) has a unique number assigned to it. Morphologicalfeatures, such as area and shape, of the blobs are used to differentiateblobs likely to be cells from those that are considered artifacts. Theuser pre-sets the morphological selection criteria by either typing inknown cell morphological features or by using the interactive trainingutility. If objects of interest are found in the field, images areacquired for all other active channels 108, otherwise the stage isadvanced to the next field 109 in the current well. Each object ofinterest is located in the image for further analysis 110. The softwaredetermines if the object meets the criteria for a valid cell nucleus 111by measuring its morphological features (size and shape). For each validcell, the XYZ stage location is recorded, a small image of the cell isstored, and features are measured 112.

The cell scanning method of the present invention can be used to performmany different assays on cellular samples by applying a number ofanalytical methods simultaneously to measure features at multiplewavelengths. An example of one such assay provides for the followingmeasurements:

1. The total fluorescent intensity within the cell nucleus for colors1-4

2. The area of the cell nucleus for color 1 (the primary marker)

3. The shape of the cell nucleus for color 1 is described by three shapefeatures:

a) perimeter squared area

b) box area ratio

c) height width ratio

4. The average fluorescent intensity within the cell nucleus for colors1-4 (i.e. #1 divided by #2)

5. The total fluorescent intensity of a ring outside the nucleus (seeFIG. 10) that represents fluorescence of the cell's cytoplasm(cytoplasmic mask) for colors 2-4

6. The area of the cytoplasmic mask

7. The average fluorescent intensity of the cytoplasmic mask for colors2-4 (i.e. #5 divided by #6)

8. The ratio of the average fluorescent intensity of the cytoplasmicmask to average fluorescent intensity within the cell nucleus for colors2-4 (i.e. #7 divided by #4)

9. The difference of the average fluorescent intensity of thecytoplasmic mask and the average fluorescent intensity within the cellnucleus for colors 2-4 (i.e. #7 minus #4)

10. The number of fluorescent domains (also call spots, dots, or grains)within the cell nucleus for colors 2-4

Features 1 through 4 are general features of the different cellscreening assays of the invention. These steps are commonly used in avariety of image analysis applications and are well known in art (Russ(1992) The Image Processing Handbook, CRC Press Inc.; Gonzales et al.(1987), Digital Image Processing. Addison-Wesley Publishing Co. pp.391-448). Features 5-9 have been developed specifically to providemeasurements of a cell's fluorescent molecules within the localcytoplasmic region of the cell and the translocation (i.e. movement) offluorescent molecules from the cytoplasm to the nucleus. These features(steps 5-9) are used for analyzing cells in microplates for theinhibition of nuclear translocation. For example, inhibition of nucleartranslocation of transcription factors provides a novel approach toscreening intact cells (detailed examples of other types of screens willbe provided below). A specific algorithm measures the amount of probe inthe nuclear region (feature 4) versus the local cytoplasmic region(feature 7) of each cell. Quantification of the difference between thesetwo sub-cellular compartments provides a measure of cytoplasm-nucleartranslocation (feature 9).

Feature 10 describes a screen used for counting of DNA or RNA probeswithin the nuclear region in colors 2-4. For example, probes arecommercially available for identifying chromosome-specific DNA sequences(Life Technologies, Gaithersburg, Md.; Genosys, Woodlands, Tex.;Biotechnologies, Inc., Richmond, Calif.; Bio 101, Inc., Vista, Calif.)Cells are three-dimensional in nature and when examined at a highmagnification under a microscope one probe may be in-focus while anothermay be completely out-of-focus. The cell screening method of the presentinvention provides for detecting three-dimensional probes in nuclei byacquiring images from multiple focal planes. The software moves theZ-axis motor drive 5 (FIG. 1) in small steps where the step distance isuser selected to account for a wide range of different nucleardiameters. At each of the focal steps, an image is acquired. The maximumgray-level intensity from each pixel in each image is found and storedin a resulting maximum projection image. The maximum projection image isthen used to count the probes. The above algorithm works well incounting probes that are not stacked directly above or below anotherone. To account for probes stacked on top of each other in theZ-direction, users can select an option to analyze probes in each of thefocal planes acquired. In this mode, the scanning system performs themaximum plane projection algorithm as discussed above, detects proberegions of interest in this image, then further analyzes these regionsin all the focal plane images.

After measuring cell features 112 (FIG. 9), the system checks if thereare any unprocessed objects in the current field 113. If there are anyunprocessed objects, it locates the next object 110 and determineswhether it meets the criteria for a valid cell nucleus 111, and measuresits features. Once all the objects in the current field are processed,the system determines whether analysis of the current plate is complete114; if not, it determines the need to find more cells in the currentwell 115. If the need exists, the system advances the XYZ stage to thenext field within the current well 109 or advances the stage to the nextwell 116 of the plate.

After a plate scan is complete, images and data can be reviewed with thesystem's image review, data review, and summary review facilities. Allimages, data, and settings from a scan are archived in the system'sdatabase for later review or for interfacing with a network informationmanagement system. Data can also be exported to other third-partystatistical packages to tabulate results and generate other reports.Users can review the images alone of every cell analyzed by the systemwith an interactive image review procedure 117. The user can review dataon a cell-by-cell basis using a combination of interactive graphs, adata spreadsheet of measured features, and images of all thefluorescence channels of a cell of interest with the interactivecell-by-cell data review procedure 118. Graphical plotting capabilitiesare provided in which data can be analyzed via interactive graphs suchas histograms and scatter plots. Users can review summary data that areaccumulated and summarized for all cells within each well of a platewith an interactive well-by-well data review procedure 119. Hard copiesof graphs and images can be printed on a wide range of standardprinters.

As a final phase of a complete scan, reports can be generated on one ormore statistics of the measured features. Users can generate a graphicalreport of data summarized on a well-by-well basis for the scanned regionof the plate using an interactive report generation procedure 120. Thisreport includes a summary of the statistics by well in tabular andgraphical format and identification information on the sample. Thereport window allows the operator to enter comments about the scan forlater retrieval. Multiple reports can be generated on many statisticsand be printed with the touch of one button. Reports can be previewedfor placement and data before being printed.

The above-recited embodiment of the method operates in a single highresolution mode referred to as the high content screening (HCS) mode.The HCS mode provides sufficient spatial resolution within a well (onthe order of 1 μm) to define the distribution of material within thewell, as well as within individual cells in the well. The high degree ofinformation content accessible in that mode, comes at the expense ofspeed and complexity of the required signal processing.

In an alternative embodiment, a high throughput system (HTS) is directlycoupled with the HCS either on the same platform or on two separateplatforms connected electronically (e.g. via a local area network). Thisembodiment of the invention, referred to as a dual mode optical system,has the advantage of increasing the throughput of an HCS by coupling itwith an HTS and thereby requiring slower high resolution dataacquisition and analysis only on the small subset of wells that show aresponse in the coupled HTS.

High throughput ‘whole plate’ reader systems are well known in the artand are commonly used as a component of an HTS system used to screenlarge numbers of compounds (Beggs et al. (1997), supra; McCaffrey et al.(1996), supra). The HTS of the present invention is carried out on themicrotiter plate or microwell array by reading many or all wells in theplate simultaneously with sufficient resolution to make determinationson a well-by-well basis. That is, calculations are made by averaging thetotal signal output of many or all the cells or the bulk of the materialin each well. Wells that exhibit some defined response in the HTS (the‘hits’) are flagged by the system. Then on the same microtiter plate ormicrowell array, each well identified as a hit is measured via HCS asdescribed above. Thus, the dual mode process involves:

1. Rapidly measuring numerous wells of a microtiter plate or microwellarray,

2. Interpreting the data to determine the overall activity offluorescently labeled reporter molecules in the cells on a well-by-wellbasis to identify “hits” (wells that exhibit a defined response),

3. Imaging numerous cells in each “hit” well, and

4. Interpreting the digital image data to determine the distribution,environment or activity of the fluorescently labeled reporter moleculesin the individual cells (i.e. intracellular measurements) and thedistribution of the cells to test for specific biological functions

In a preferred embodiment of dual mode processing (FIG. 11), at thestart of a run 301, the operator enters information 302 that describesthe plate and its contents, specifies the filter settings andfluorescent channels to match the biological labels being used, theinformation sought and the camera settings to match the samplebrightness. These parameters are stored in the system's database foreasy retrieval for each automated run. The microtiter plate or microwellarray is loaded into the cell screening system 303 either manually orautomatically by controlling a robotic loading device. An optionalenvironmental chamber 304 is controlled by the system to maintain thetemperature, humidity and CO₂ levels in the air surrounding live cellsin the microtiter plate or microwell array. An optional fluid deliverydevice 305 (see FIG. 8) is controlled by the system to dispense fluidsinto the wells during the scan.

High throughput processing 306 is first performed on the microtiterplate or microwell array by acquiring and analyzing the signal from eachof the wells in the plate. The processing performed in high throughputmode 307 is illustrated in FIG. 12 and described below. Wells thatexhibit some selected intensity response in this high throughput mode(“hits”) are identified by the system. The system performs a conditionaloperation 308 that tests for hits. If hits are found, those specific hitwells are further analyzed in high content (micro level) mode 309. Theprocessing performed in high content mode 312 is illustrated in FIG. 13.The system then updates 310 the informatics database 311 with results ofthe measurements on the plate. If there are more plates to be analyzed313 the system loads the next plate 303; otherwise the analysis of theplates terminates 314.

The following discussion describes the high throughput mode illustratedin FIG. 12. The preferred embodiment of the system, the single platformdual mode screening system, will be described. Those skilled in the artwill recognize that operationally the dual platform system simplyinvolves moving the plate between two optical systems rather than movingthe optics. Once the system has been set up and the plate loaded, thesystem begins the HTS acquisition and analysis 401. The HTS opticalmodule is selected by controlling a motorized optical positioning device402 on the dual mode system. In one fluorescence channel, data from aprimary marker on the plate is acquired 403 and wells are isolated fromthe plate background using a masking procedure 404. Images are alsoacquired in other fluorescence channels being used 405. The region ineach image corresponding to each well 406 is measured 407. A featurecalculated from the measurements for a particular well is compared witha predefined threshold or intensity response 408, and based on theresult the well is either flagged as a “hit” 409 or not. The locationsof the wells flagged as hits are recorded for subsequent high contentmode processing. If there are wells remaining to be processed 410 theprogram loops back 406 until all the wells have been processed 411 andthe system exits high throughput mode.

Following HTS analysis, the system starts the high content modeprocessing 501 defined in FIG. 13. The system selects the HCS opticalmodule 502 by controlling the motorized positioning system. For each“hit” well identified in high throughput mode, the XY stage location ofthe well is retrieved from memory or disk and the stage is then moved tothe selected stage location 503. The autofocus procedure 504 is calledfor the first field in each hit well and then once every 5 to 8 fieldswithin each well. In one channel, images are acquired of the primarymarker 505 (typically cell nuclei counterstained with DAPI, Hoechst orPI fluorescent dye). The images are then segmented (separated intoregions of nuclei and non-nuclei) using an adaptive thresholdingprocedure 506. The output of the segmentation procedure is a binary maskwherein the objects are white and the background is black. This binaryimage, also called a mask in the art, is used to determine if the fieldcontains objects 507. The mask is labeled with a blob labeling algorithmwhereby each object (or blob) has a unique number assigned to it. Ifobjects are found in the field, images are acquired for all other activechannels 508, otherwise the stage is advanced to the next field 514 inthe current well. Each object is located in the image for furtheranalysis 509. Morphological features, such as area and shape of theobjects, are used to select objects likely to be cell nuclei 510, anddiscard (do no further processing on) those that are consideredartifacts. For each valid cell nucleus, the XYZ stage location isrecorded, a small image of the cell is stored, and assay specificfeatures are measured 511. The system then performs multiple tests onthe cells by applying several analytical methods to measure features ateach of several wavelengths. After measuring the cell features, thesystems checks if there are any unprocessed objects in the current field512. If there are any unprocessed objects, it locates the next object509 and determines whether it meets the criteria for a valid cellnucleus 510, and measures its features. After processing all the objectsin the current field, the system determines whether it needs to findmore cells or fields in the current well 513. If it needs to find morecells or fields in the current well it advances the XYZ stage to thenext field within the current well 515. Otherwise, the system checkswhether it has any remaining hit wells to measure 515. If so, itadvances to the next hit well 503 and proceeds through another cycle ofacquisition and analysis, otherwise the HCS mode is finished 516.

In an alternative embodiment of the present invention, a method ofkinetic live cell screening is provided. The previously describedembodiments of the invention are used to characterize the spatialdistribution of cellular components at a specific point in time, thetime of chemical fixation. As such, these embodiments have limitedutility for implementing kinetic based screens, due to the sequentialnature of the image acquisition, and the amount of time required to readall the wells on a plate. For example, since a plate can require 30-60minutes to read through all the wells, only very slow kinetic processescan be measured by simply preparing a plate of live cells and thenreading through all the wells more than once. Faster kinetic processescan be measured by taking multiple readings of each well beforeproceeding to the next well, but the elapsed time between the first andlast well would be too long, and fast kinetic processes would likely becomplete before reaching the last well.

The kinetic live cell extension of the invention enables the design anduse of screens in which a biological process is characterized by itskinetics instead of, or in addition to, its spatial characteristics. Inmany cases, a response in live cells can be measured by adding a reagentto a specific well and making multiple measurements on that well withthe appropriate timing. This dynamic live cell embodiment of theinvention therefore includes apparatus for fluid delivery to individualwells of the system in order to deliver reagents to each well at aspecific time in advance of reading the well. This embodiment therebyallows kinetic measurements to be made with temporal resolution ofseconds to minutes on each well of the plate. To improve the overallefficiency of the dynamic live cell system, the acquisition controlprogram is modified to allow repetitive data collection from sub-regionsof the plate, allowing the system to read other wells between the timepoints required for an individual well.

FIG. 8 describes an example of a fluid delivery device for use with thelive cell embodiment of the invention and is described above. Thisset-up allows one set of pipette tips 705, or even a single pipette tip,to deliver reagent to all the wells on the plate. The bank of syringepumps 701 can be used to deliver fluid to 12 wells simultaneously, or tofewer wells by removing some of the tips 705. The temporal resolution ofthe system can therefore be adjusted, without sacrificing datacollection efficiency, by changing the number of tips and the scanpattern as follows. Typically, the data collection and analysis from asingle well takes about 5 seconds. Moving from well to well and focusingin a well requires about 5 seconds, so the overall cycle time for a wellis about 10 seconds. Therefore, if a single pipette tip is used todeliver fluid to a single well, and data is collected repetitively fromthat well, measurements can be made with about 5 seconds temporalresolution. If 6 pipette tips are used to deliver fluids to 6 wellssimultaneously, and the system repetitively scans all 6 wells, each scanwill require 60 seconds, thereby establishing the temporal resolution.For slower processes which only require data collection every 8 minutes,fluids can be delivered to one half of the plate, by moving the plateduring the fluid delivery phase, and then repetitively scanning thathalf of the plate. Therefore, by adjusting the size of the sub-regionbeing scanned on the plate, the temporal resolution can be adjustedwithout having to insert wait times between acquisitions. Because thesystem is continuously scanning and acquiring data, the overall time tocollect a kinetic data set from the plate is then simply the time toperform a single scan of the plate, multiplied by the number of timepoints required. Typically, 1 time point before addition of compoundsand 2 or 3 time points following addition should be sufficient forscreening purposes.

FIG. 14 shows the acquisition sequence used for kinetic analysis. Thestart of processing 801 is configuration of the system, much of which isidentical to the standard HCS configuration. In addition, the operatormust enter information specific to the kinetic analysis being performed802, such as the sub-region size, the number of time points required,and the required time increment. A sub-region is a group of wells thatwill be scanned repetitively in order to accumulate kinetic data. Thesize of the sub-region is adjusted so that the system can scan a wholesub-region once during a single time increment, thus minimizing waittimes. The optimum sub-region size is calculated from the setupparameters, and adjusted if necessary by the operator. The system thenmoves the plate to the first sub-region 803 and to the first well inthat sub-region 804 to acquire the prestimulation (time=0) time points.The acquisition sequence performed in each well is exactly the same asthat required for the specific HCS being run in kinetic mode. FIG. 15details a flow chart for that processing. All of the steps between thestart 901 and the return 902 are identical to those described as steps504-514 in FIG. 13.

After processing each well in a sub-region, the system checks to see ifall the wells in the sub-region have been processed 806 (FIG. 14), andcycles through all the wells until the whole region has been processed.The system then moves the plate into position for fluid addition, andcontrols fluidic system delivery of fluids to the entire sub-region 807.This may require multiple additions for sub-regions which span severalrows on the plate, with the system moving the plate on the X,Y stagebetween additions. Once the fluids have been added, the system moves tothe first well in the sub-region 808 to begin acquisition of timepoints. The data is acquired from each well 809 and as before the systemcycles through all the wells in the sub-region 810. After each passthrough the sub-region, the system checks whether all the time pointshave been collected 811 and if not, pauses 813 if necessary 812 to staysynchronized with the requested time increment. Otherwise, the systemchecks for additional sub-regions on the plate 814 and either moves tothe next sub-region 803 or finishes 815. Thus, the kinetic analysis modecomprises operator identification of sub-regions of the microtiter plateor microwells to be screened, based on the kinetic response to beinvestigated, with data acquisitions within a sub-region prior to dataacquisition in subsequent sub-regions.

Specific Screens

In another aspect of the present invention, a machine readable storagemedium comprising a program containing a set of instructions for causinga cell screening system to execute procedures for defining thedistribution and activity of specific cellular constituents andprocesses is provided. In a preferred embodiment, the cell screeningsystem comprises a high magnification fluorescence optical system with astage adapted for holding cells and a means for moving the stage, adigital camera, a light source for receiving and processing the digitaldata from the digital camera, and a computer means for receiving andprocessing the digital data from the digital camera. This aspect of theinvention comprises programs that instruct the cell screening system todefine the distribution and activity of specific cellular constituentsand processes, using the luminescent probes, the optical imaging system,and the pattern recognition software of the invention. Preferredembodiments of the machine readable storage medium comprise programsconsisting of a set of instructions for causing a cell screening systemto execute the procedures set forth in FIG. 9, 11, 12, 13, 14, 15, or28. Another preferred embodiment comprises a program consisting of a setof instructions for causing a cell screening system to executeprocedures for detecting the distribution and activity of specificcellular constituents and processes. In most preferred embodiments, thecellular processes include, but are not limited to, nucleartranslocation of a protein, cellular hypertrophy, apoptosis,transmembrane receptor internalization, and protease-inducedtranslocation of a protein.

The following examples are intended for purposes of illustration onlyand should not be construed to limit the scope of the invention, asdefined in the claims appended hereto.

The various chemical compounds, reagents, dyes, and antibodies that arereferred to in the following Examples are commercially available fromsuch sources as Sigma Chemical (St. Louis, Mo.), Molecular Probes(Eugene, Oreg.), Aldrich Chemical Company (Milwaukee, Wis.), AccurateChemical Company (Westbury, N.Y.), Jackson Immunolabs, and Clontech(Palo Alto, Calif.).

EXAMPLE 1 Automated Screen for Compounds that Induce or Inhibit NuclearTranslocation of a DNA Transcription Factor

Regulation of transcription of some genes involves activation of atranscription factor in the cytoplasm, resulting in that factor beingtransported into the nucleus where it can initiate transcription of aparticular gene or genes. This change in transcription factordistribution is the basis of a screen for the cell-based screeningsystem to detect compounds that inhibit or induce transcription of aparticular gene or group of genes. A general description of the screenis given followed by a specific example.

The distribution of the transcription factor is determined by labelingthe nuclei with a DNA specific fluorophore like Hoechst 33423 and thetranscription factor with a specific fluorescent antibody. Afterautofocusing on the Hoechst labeled nuclei, an image of the nuclei isacquired in the cell-based screening system at 20× magnification andused to create a mask by one of several optional thresholding methods,as described supra. The morphological descriptors of the regions definedby the mask are compared with the user defined parameters and validnuclear masks are identified and used with the following algorithm toextract transcription factor distributions. Each valid nuclear mask iseroded to define a slightly smaller nuclear region. The original nuclearmask is then dilated in two steps to define a ring shaped region aroundthe nucleus, which represents a cytoplasmic region. The average antibodyfluorescence in each of these two regions is determined, and thedifference between these averages is defined as the NucCyt Difference.Two examples of determining nuclear translocation are discussed belowand illustrated in FIGS. 10A-J. FIG. 10A illustrates an unstimulatedcell with its nucleus 200 labeled with a blue fluorophore and atranscription factor in the cytoplasm 201 labeled with a greenfluorophore. FIG. 10B illustrates the nuclear mask 202 derived by thecell-based screening system. FIG. 10C illustrates the cytoplasm 203 ofthe unstimulated cell imaged at a green wavelength. FIG. 10D illustratesthe nuclear mask 202 is eroded (reduced) once to define a nuclearsampling region 204 with minimal cytoplasmic distribution. The nucleusboundary 202 is dilated (expanded) several times to form a ring that is2-3 pixels wide that is used to define the cytoplasmic sampling region205 for the same cell. FIG. 10E further illustrates a side view whichshows the nuclear sampling region 204 and the cytoplasmic samplingregion 205. Using these two sampling regions, data on nucleartranslocation can be automatically analyzed by the cell-based screeningsystem on a cell by cell basis. FIGS. 10F-J illustrates the strategy fordetermining nuclear translocation in a stimulated cell. FIG. 10Fillustrates a stimulated cell with its nucleus 206 labeled with a bluefluorophore and a transcription factor in the cytoplasm 207 labeled witha green fluorophore. The nuclear mask 208 in FIG. 10G is derived by thecell based screening system. FIG. 10H illustrates the cytoplasm 209 of astimulated cell imaged at a green wavelength. FIG. 10I illustrates thenuclear sampling region 211 and cytoplasmic sampling region 212 of thestimulated cell. FIG. 10J further illustrates a side view which showsthe nuclear sampling region 211 and the cytoplasmic sampling region 212.

A specific application of this method has been used to validate thismethod as a screen. A human cell line was plated in 96 well microtiterplates. Some rows of wells were titrated with agonist, a known inducerof a specific nuclear transcription factor. The cells were then fixedand stained by standard methods with a fluorescein labeled antibody tothe transcription factor, and Hoechst 33423. The cell-based screeningsystem was used to acquire and analyze images from this plate and theNucCyt Difference was found to be strongly correlated with the amount ofagonist added to the wells as illustrated in FIG. 16. In a secondexperiment, an antagonist to the receptor for the agonist was titratedin the presence of agonist, progressively inhibiting agonist-inducedtranslocation of the transcription factor. The NucCyt Difference wasfound to strongly correlate with this inhibition of translocation, asillustrated in FIG. 17.

Additional experiments have shown that the NucCyt Difference givesconsistent results over a wide range of cell densities and reagentconcentrations, and can therefore be routinely used to screen compoundlibraries for specific nuclear translocation activity. Furthermore, thesame method can be used with antibodies to other transcription factors,or GFP-transcription factor chimeras, in living and fixed cells, toscreen for effects on the regulation of transcription of this and othergenes.

FIG. 18 is a representative display on a PC screen of data which wasobtained in accordance with Example 1. Graph 1 180 plots the differencebetween the average antibody fluorescence in the nuclear sampling regionand cytoplasmic sampling region, NucCyt Difference verses Well #. Graph2 181 plots the average fluorescence of the antibody in the nuclearsampling region, NP1 average, versus the Well #. Graph 3 182 plots theaverage antibody fluorescence in the cytoplasmic sampling region, LIP1average, versus Well #. The software permits displaying data from eachcell. For example, FIG. 18 shows a screen display 183, the nuclear image184, and the fluorescent antibody image 185 for cell #26.

NucCyt Difference referred to in graph 1 180 of FIG. 18 is thedifference between the average cytoplasmic probe (fluorescent reportermolecule) intensity and the average nuclear probe (fluorescent reportermolecule) intensity. NP1 average referred to in graph 2 181 of FIG. 18is the average of cytoplasmic probe (fluorescent reporter molecule)intensity within the nuclear sampling region. LIP1 average referred toin graph 3 182 of FIG. 18 is the average probe (fluorescent reportermolecule) intensity within the cytoplasmic sampling region.

EXAMPLE 2 Automated Screen for Compounds that Induce or InhibitHypertrophy in Cardiac Myocytes

Hypertrophy in cardiac myocytes has been associated with a cascade ofalterations in gene expression and can be characterized in cell cultureby an alteration in cell size, that is clearly visible in adherent cellsgrowing on a coverslip. A screen is implemented using the followingstrategy. Myocyte cell line QM7 (Quail muscle clone 7; ATCC CRL-1962)cultured in 96 well plates, can be treated with various compounds andthen fixed and labeled with a fluorescent antibody to a cell surfacemarker and a DNA label like Hoechst. After focusing on the Hoechstlabeled nuclei, two images are acquired, one of the Hoechst labelednuclei and one of the fluorescent antibody. The nuclei are identified bythresholding to create a mask and then comparing the morphologicaldescriptors of the mask with a set of user defined descriptor values.Local regions containing cells are defined around the nuclei. The limitsof the cells in those regions are then defined by a local dynamicthreshold operation on the same region in the fluorescent antibodyimage. A sequence of erosions and dilations is used to separate slightlytouching cells and a second set of morphological descriptors is used toidentify single cells. The area of the individual cells is tabulated inorder to define the distribution of cell sizes for comparison with sizedata from normal and hypertrophic cells. In addition, a secondfluorescent antibody to a particular cellular protein, such as one ofthe major muscle proteins actin or myosin can be included. Images ofthis second antibody can be acquired and stored with the above images,for later review, to identify anomalies in the distribution of theseproteins in hypertrophic cells, or algorithms can be developed toautomatically analyze the distributions of the labeled proteins in theseimages.

EXAMPLE 3 Automated Screens for Compounds that Induce or InhibitReceptor Internalization

G-protein Coupled Receptors

G-protein coupled receptors (GPCRs) are a large class of 7transmembrane-domain cell surface receptors that transmit signals fromthe extracellular environment to the cell cytoplasm via theirinteraction with heterotrimeric G-proteins. Activation of thesereceptors by ligand binding promotes the exchange of GDP for GTP on theassociated G-protein, resulting in the dissociation of the G-proteininto active Gα-GTP and Gβγ subunits. The interaction of these subunitswith their effectors stimulates a cascade of secondary signals in thecell, such as the production of cyclic AMP (cAMP) and inositoltriphosphate (IP₃), Ca⁺⁺ mobilization, and activation of a variety ofkinases. A wide range of biological functions are associated with GPCRs,including, but not limited to, smell, taste, perception of light,control of blood pressure, neurotransmission, endocrine and exocrinefunction, chemotaxis, exocytosis, embryogenesis and development, cellgrowth and differentiation, and oncogenesis. GPCRs have therefore becomea major potential target for a variety of therapeutic units.

GPCRs span the plasma membrane and undergo a relatively slow rate ofendocytosis from the cell surface to endosomes in unstimulated cells.Although poorly understood mechanistically, it is known that thepresence of agonist increases the rate of receptor internalizationdramatically. Once internalized in endosomes, GPCRs may either berecycled back to the plasma membrane or targeted to lysosomes fordegradation. The significance of this sequestration of GPCRs is not yetfully understood. Receptor internalization may play a role indesensitization (loss of functional response) exhibited as a reductionin the ability of the receptor to generate second messenger in thepresence of continued stimulation. However, the rate of receptor lossfrom the surface is usually too slow to account for the rapid rate ofdesensitization (Tobin, A. B. et al. (1992) Mol. Pharmacol. 42:1042-1048), and there are examples where the two processes have beenshown to be uncoupled (Baumgold, J. et al. (1989) Neuropharmacology 28:1253-1261; Kanbe, S. et al. (1990) Biochem. Pharmacol. 40: 1005-1014).

It is likely that endocytosis of receptors may be involved inresensitization (the reestablishment of the ability of the cell toproduce second messenger in response to stimulation). It has beendemonstrated for the β₂-adrenergic receptor (β₂-AR) that sequestrationdeficient mutants as well as receptors treated with agents that blocksequestration do not resensitize (Yu, S. S. et al. (1993) J. Biol. Chem.268(1): 337-341; Barak, L. S. et al. (1994) J. Biol. Chem. 269(4):2790-2795). For the β₂-AR, agonist stimulation results in receptorphosphorylation by protein kinase A and β₂-adrenergic receptor kinase(β-ARK). Subsequently there is uncoupling of the receptor from its Gprotein as a result of the recruitment and binding of β-arrestin to thereceptor, and internalization of the receptor via clathrin-coated pitsis initiated. The acidic endosomal pH favors phosphatase activity, thusenhancing receptor dephosphorylation (Krueger, K. M. et al. (1997) J.Biol. Chem. 272(1): 5-8) and making the receptor available for recyclingto the plasma membrane to reassociate with a G-protein. However,resensitization of other receptors, such as the M₄ muscarinic receptors,has been shown to be delayed by endocytosis (Bogatekewitsch, G. S. etal. (1996) Mol. Pharmacol. 50: 424-429). Despite the fact that thefunctional importance of receptor internalization may vary betweenreceptor classes, it remains clear that internalization is a significantstep in the pathway of receptor activation and function.

The fundamental importance of cellular processes involving GPCRs makesthem a significant target for drug screening. The state of the art formonitoring GPCR-ligand interactions and receptor internalization islimited to measurements of a single event (e.g., receptor-ligandinteraction or receptor loss from the plasma membrane). Currentprocedures include measurements of binding of labeled ligand (usuallyradioactively labeled) to whole cells or isolated membrane fractions(WO/97/04214; von Zastrow and Kobilka, J. Biol. Chem. 269:18448-18452(1994); Koch et al., J. Biol. Chem. 273:13652-13657 (1998); Tiberi etal., J. Biol. Chem. 271:3771-3778 (1996)), the coincident migration ofreceptors with various markers into subcellular fractions resolvedthrough centrifugation (Seibold et al., J. Biol. Chem. 273:7637-7642(1998); Stefan et al., Mol. Biol. Cell. 9:885-899 (1998)); visualizationof fluorescently labeled ligand binding to receptors in fixed cells(Tarasova et al., J. Biol. Chem. 272:14817-14824 (1997)), or antibodylabeling (either directly or to epitope tags) to identify receptors (vonZastrow and Kobilka, J. Biol. Chem. 269:18448-18452 (1994); Segredo etal. (1997) J. Neurochem. 68: 2395-2404; Krueger et al. (1997) J. Biol.Chem. 272(1): 5-8; Tiberi et al. (1996) J. Biol. Chem. 271(7):3771-3778)). More recently, green fluorescent protein (GFP)-receptorfusions have been used, which allows visualization of GPCR receptortrafficking in live cells (Kallal, L. et al. (1998) J. Biol. Chem.273(1): 322-328; Tarasova, N. I. et al. (1997) J. Biol. Chem. 272(23):14817-14824). However, this requires confocal imaging to obtainthree-dimensional information in order to distinguish whether a receptorhas been internalized or has simply moved in the plane of the plasmamembrane. Methods have also been disclosed for the identification ofGPCRs, their ligands, and compounds that modulate their activity (WO98/13353 and WO 97/48820). These methods, however, detect G-proteinactivation indirectly by ligand binding to the receptor and reportergene activation. Neither method directly labels the receptor or directlymeasures the internalization of the receptor as an indication ofreceptor activation.

While existing approaches have provided information and a means ofmeasuring receptor function, there remains a need in the art for amethod to directly measure ligand-induced receptor internalization withhigh spatial and temporal resolution as a measure of receptoractivation.

Therefore, a novel approach to measuring receptor internalization isdescribed here that permits the measurement of receptor internalizationin a single step with appropriate automation and throughput. Thisapproach involves fluorescent labeling of the GPCR and the automatedmeasurement of GPCR internalization in stimulated cells. An alternativenovel approach described here involves using dual labeled receptors,comprising a label specific to the amino terminus of the receptor todistinctly label its extracellular domain in addition to amolecular-based chromophore such as GFP or luciferase on the receptor'scarboxy terminus to specifically label the intracellular domain. Methodsfor the construction of such chimeric protein-expressing DNA constructsare well known in the art. (Molecular Cloning: A Laboratory Manual(Sambrook, et al., 1989, Cold Spring Harbor Laboratory Press), GeneExpression Technology (Methods in Enzymology, Vol. 185, edited by D.Goeddel, 1991. Academic Press, San Diego, Calif.); PCR Protocols: AGuide to Methods and Applications (Innis, et al. 1990. Academic Press,San Diego, Calif.); Gene Transfer and Expression Protocols, pp. 109-128,ed. E. J. Murray, The Humana Press Inc., Clifton, N.J.).

A ratio of fluorescence intensity of the two labels is made inunstimulated and stimulated cells. Since the amino terminus of thereceptor is only available for labeling while the receptor is insertedin the plasma membrane, the ratio of the two labels in unpermeabilizedcells can be used to measure the extent of internalization of thereceptor. There is currently no known technology for simultaneouslymeasuring the relative extracellular availability of external andinternal domains of membrane receptors.

In a preferred embodiment of the screen for modulaters of GPCRs, livingcells are obtained from continuous lines of normal or transformed cells,or primary normal or transformed cells obtained directly from animals.The appropriate cells may be transiently or stably transfected with aDNA construct (either plasmid or viral based) that expresses the GPCR ofinterest fused to a molecular based chromophore at either its amino orcarboxy terminus or internally such that the receptor retains function.Examples of useful molecular-based chromophores include, but are notlimited to, GFP and any of its various mutants (Heim and Tsien (1996)Current Biology 6: 178-182; Zhang et al. (1996) Biochem. Biophys. Res.Comm. 227: 707-711). In addition, any of the luciferases and theirmutants could also be used. This would be a novel labeling techniquesince the examples of use of this molecular-based chromophore to datehave included use as a reporter of gene activity (Yang et al. (1998) J.Biol. Chem. 273(17): 10763-10770; Peng et al. (1998) J. Biol. Chem.273(27): 17286-17295; Baldari et al. (1998) Biologicals 26(1): 1-5)) andconstruction of biosensors (Campbell and Patel (1983) Biochem. J. 216:185-194; Sala-Newby and Campbell (1992) FEBS Lett. 307: 241-244; Jenkinset al. (1990) Biochem. Soc. Trans. 18: 463-464) but not as a chimerasfor marking a particular protein target. The expression of theGPCR-luminescent protein fusion may be constitutive (driven by any of avariety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive).

Alternatively, the cells are transiently or stably transfected with aDNA construct (either plasmid or viral based) that expresses the GPCR ofinterest fused to a small peptide or epitope tag. The epitope tag may befused to the amino or carboxy terminus, or internally such that thereceptor remains functional, or, alternatively, the GPCR may be labeledwith two distinct epitope tags, with one being fused to each end of theGPCR. Some examples of epitope tags include, but are not limited to,FLAG (Sigma Chemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad,Calif.), 6-His (Invitrogen; Novagen, Madison, Wis.), and HA (BoehringerManheim Biochemicals). The expression the GPCR fusion may beconstitutive or inducible.

In another embodiment, the cells are transiently or stably transfectedwith a DNA construct (either plasmid or viral based) that expresses theGPCR of interest fused to an epitope tag at its amino terminus and amolecular based chromophore at its carboxy terminus. Alternatively, theGPCR may be fused to an epitope tag at its carboxyl terminus and amolecular based chromophore at its amino terminus. The expression of theGPCR fusion may be constitutive or inducible.

The appropriate cells are then patterned into arrays for treatment andanalysis. These arrays can be multiple well plates containing 96, 384,1536, or more individual wells. The cells can also be arranged intomicroarrays of “virtual wells” such as those described in U.S. Pat. No.6,103,479. These microarrrays can be of the same cell type and aretreated with a combinatorial of distinct compounds, or alternatively,the microarrrays can be a combinatorial of cell types treated with oneor more compounds.

Once the chosen cells are patterned into wells or microarrays, they aretreated with solutions of drugs or ligands to either inhibit orstimulate receptor internalization. The fluidic delivery system can bemanual, robotic, or the microfluidics of the CELLCHIP™ system (U.S. Pat.No. 6,103,479). After an appropriate incubation period, the cells arefixed with a chemical crosslinking agent and stained withluminescence-based reagents. These reagents include, but are not limitedto, luminescently labeled primary or secondary antibodies that reactwith the GPCR, the epitope tag, or other cellular antigens determined tocorrelate with internalization of the GPCR. Luminescent stains, dyes,and other small molecules can also be used to measure the physiologicalresponse of the cells to drugs. These reagents are used to measure thetemporal and spatial changes in ions, metabolites, macromolecules, andorganelles induced by drugs. Macromolecular-based indicators of cellularphysiology can also be used in the assay.

In another embodiment, cells in wells or microarrays are treated withdrugs, and the physiological response is measured temporally andspatially within a population of single living cells after anappropriate incubation period. Luminescent stains, dyes, and other smallmolecules can be used to measure the physiological response of livingcells to drugs. Molecular-based chromophores expressed by the cellsthemselves (such as GFP and its mutants) are particularly suited to livecell measurements. These reagents can be used to measure the temporaland spatial intracellular changes of ions, metabolites, macromolecules,and organelles induced by drugs. Macromolecular-based indicators ofcellular physiology can also be used in the assay. These luminscentanalogs and biosensors can be used to measure the temporal and spatialchanges in the distribution and activity of macromolecules such asprotein, DNA, RNA, lipids, and carbohydrates in response to drugtreatments.

In another embodiment, fluorescently labeled ligand is used to inducereceptor sequestration and the fate of the ligand is following as aparameter of the high-content screen.

In another embodiment, cells may contain more than one distinctlylabeled receptor such that different GPCRs can be analyzed in the samecells by using different fluorescence channels to collect those data.Similarly, the wells or microarrays may contain mixed populations ofcells, each population containing a different receptor labeled with aspectrally distinct fluorophore. It is possible to measure the effectsof drugs on different receptors in a single run by utilizing a cellscreening system, such as the cell screening system of the presentinvention, that is capable of distinguishing the channels offluorescence of the different receptors in these examples. In this wayone can screen for compounds that affect multiple receptor types or,conversely, for compounds that affect one receptor type and not others.

It will be obvious to one skilled in the art that this invention can beapplied to any cell surface receptor that undergoes internalization inresponse to agonist stimulation. Some known examples of GPCRs are theadrenergic receptors; muscarinic acetylcholine receptors; opioidreceptors; chemokine receptors; neuropeptide receptors; prostaglandinreceptors; parathyroid hormone receptor; cholecystokinin receptor;secretin receptor; rhodopsin; dopamine receptors; serotonin receptors;odorant receptors; histamine receptors; angiotensin receptors; gastrinreceptors; follicle stimulating hormone receptor; luteinizing hormonereceptor; metabotropic glutamate receptors; glucagon receptor (a morecomplete list of known GPCRs and their ligands can be found inBeck-Sickinger, A. G (1996) Drug Discovery Today 1(12): 502-513). Thisinvention is not limited to GPCRs; examples of other receptors to whichthis invention could be applied include, but are not limited to, growthfactor receptors such as PDGF (Heldin et al. (1982) J. Biol. Chem.257(8): 4216-4221; Kapeller et al. (1993) Mol. Cell. Biol. 13(10):6052-6063) and EGF (Zidovetzki et al. (1981) Proc. Natl. Acad. Sci.78(11): 6981-6985; Beguinot et al. (1984) Proc. Natl. Acad. Sci. 81(8):2384-2388; Emlet et al. (1997) J. Biol. Chem. 272(7): 4079-4086), thetransferrin receptor (Klausner et al. (1983) J. Biol. Chem. 258(8):4715-4724; Ciechanover et al. (1983) J. Cell. Biochem. 23(1-4):107-130), and the insulin receptor (Baldwin et al. (1980) Proc. Natl.Acad. Sci. 77(10): 5975-5978; Di Guglielmo et al. (1998) Mol. Cell.Biochem. 182(1-2): 59-63). This invention can also be applied to orphanreceptors for which a specific ligand and/or effector is unknown.

The following example is a screen for activation of a G-protein coupledreceptor (GPCR) as detected by the translocation of the GPCR from theplasma membrane to a proximal nuclear location. This example illustrateshow a high throughput screen can be coupled with a high-content screenin the dual mode System for Cell Based Screening.

FIG. 19 illustrates a dual mode screen for activation of a GPCR. Cellscarrying a stable chimera of the GPCR with a blue fluorescent protein(BFP) are loaded with the acetoxymethylester form of Fluo-3, a cellpermeable calcium indicator (green fluorescence) that is trapped inliving cells by the hydrolysis of the esters. They are then depositedinto the wells of a microtiter plate 601. The wells are then treatedwith an array of test compounds using a fluid delivery system, and ashort sequence of Fluo-3 images of the whole microtiter plate areacquired and analyzed for wells exhibiting a calcium response (i.e.,high throughput mode). The images appear like the illustration of themicrotiter plate 601 in FIG. 19. A small number of wells, such as wellsC4 and E9 in the illustration, would fluoresce more brightly due to theCa⁺⁺ released upon stimulation of the receptors. The locations of wellscontaining compounds that induced a response 602, would then betransferred to the HCS program and the optics switched for detailed cellby cell analysis of the blue fluorescence for evidence of GPCRtranslocation to the perinuclear region. The bottom of FIG. 19illustrates the two possible outcomes of the analysis of the highresolution cell data. The camera images a sub-region 604 of the wellarea 603, producing images of the fluorescent cells 605. In well C4, theuniform distribution of the fluorescence in the cells indicates that thereceptor has not internalized, implying that the Ca⁺⁺ response seen wasthe result of the stimulation of some other signaling system in thecell. The cells in well E9 606 on the other hand, clearly indicate aconcentration of the receptor in the perinuclear region clearlyindicating the full activation of the receptor. Because only a few hitwells have to be analyzed with high resolution, the overall throughputof the dual mode system can be quite high, comparable to the highthroughput system alone.

EXAMPLE 4 High-content Screen of Ligand-induced Parathyroid HormoneReceptor Internalization

Plasmid construct. A eukaryotic expression plasmid containing the codingsequence for a humanized GFP mutant (pEGFP-N₂, CLONTECH, Palo Alto,Calif.) was used to create a GFP-human parathyroid hormone receptor(PTHR, GenBank #L04308) chimera.

Cell preparation. The plasmid construct was used to transfect a humanembryonic kidney cell line (HEK 293) (ATCC NO.CRL-1573). Clonal linesstably expressing the GFP-PTHR chimera were established by antibioticselection with the neomycin analog Geneticin (0.5 mg/ml; LifeTechnologies, Gaithersburg, Md.). Cells are prepared and plated inDMEM/F12 medium (Life Technologies) containing 25 mM HEPES buffer (nosodium bicarbonate), 10% fetal calf serum (FCS), penicillin/streptomycin(PS), and 2 mM L-glutamine. Cells are plated at a density of 4×10⁴ perwell in a 96-well microtiter plate in a volume of 200 ul per well. Cellsare allowed to settle for approximately 30 minutes at room temperatureand the plate is then placed in a 37° C. humidified air incubator.

Parathyroid hormone induction of GFP-PTHR internalization. A 100 uMstock of bovine parathyroid hormone (PTH), amino acids 1-34 (Bachem,King of Prussia, Pa.), is prepared using acidified water (pH 4-4.5). Toinduce internalization of the GFP-PTHR chimera, cells are stimulated bythe addition of 50 ul of 500 nM PTH to each well (diluted in DMEM/F12,10% FCS, PS, 2 mM L-glutamine). This volume is added to the 200 ul ofmedium already in the well, yielding a final concentration of 100 nMPTH. The plate is incubated at room temperature for two hours whilecovered with aluminum foil to protect the fluorophore from light.Following the two hour PTH stimulation, the media is decanted from theplate and the cells are fixed and the nuclei stained by the addition of200 ul of Hank's Balanced Salt Solution (HBSS) containing 3.7% formalin(Sigma) and 1 ug/ml Hoechst 33342 (Molecular Probes, Eugene, Oreg.).After a 10 minute incubation at room temperature, the solution isdecanted from the plate, cells are washed by the addition of 200 ul/wellHBSS, and the plates are analyzed/stored with fresh HBSS (200 ul/well).

Image acquisition and analysis. (See FIG. 26 for overview) Afterautofocusing 101 (FIG. 27) on the Hoechst-labeled nuclei, an image ofthe nuclei 102 is acquired at 20× magnification. The nucleus image issegmented by thresholding 103, using a threshold value selected by theuser or obtained by one of two other methods from which the user canselect (isodata algorithm or peak interpolation). The total area inpixels of all the nuclei in the image is then computed as a single sum104. An image of the GFP fluorescence is then acquired at 20×magnification 105. (FIG. 26) The area of the plate imaged in this way iscalled a field.

Large artifacts are removed from the GFP image as follows 106. (FIG. 26)The image is thresholded at a user-selected intensity value which ishigher than the threshold used to detect valid objects later. Allobjects detected in the resulting image are labeled and their size(number of pixels) is measured. Any objects greater than auser-specified size are treated as artifacts. All such objects arecopied and pasted onto a new blank image, the artifact image. Theartifact image is dilated slightly to be sure that the artifacts will becompletely deleted; The artifact image is then subtracted from theoriginal GFP image, yielding the intermediate image.

To remove variations in the background fluorescence, the intermediateimage is subjected to a top hat transform 107. (FIG. 26) This transformconsists of (a) grayscale erosion (replacement of each pixel value bythe minimum value in its neighborhood) (b) a grayscale dilation(replacement of each pixel value by the maximum value in itsneighborhood) with the same size neighborhood as (a), producing abackground image, and (c) subtraction of the resulting background imagefrom the original input image to produce the object image, whichcontains small bright spots. The size of the neighborhood used for steps(a) and (b) above is selected to be slightly larger than all the objectsof interest in the image. As a result, all such objects are absent fromthe background image after the erosion (a) and dilation (b). However,gradual variations in the background of the original image are retainedin the background image. Therefore, the subtraction step (c) removesthese variations in the background from the object image.

The object image is processed to determine which bright spots representthe internalized receptor in stimulated cells. This process uses abrightness threshold and a minimum size set by the user. The objectimage is thresholded at the brightness threshold to create the binaryobject mask 108. (FIG. 26) The objects in the binary object mask arelabeled and their sizes are measured in pixels. Those objects that meetor exceed the minimum size are valid spots 109; (FIG. 28) the rest areignored.

The following measurements are then determined for each valid spot.(FIG. 28) The count of spots in the field is incremented 110. The numberof pixels was previously counted. For each valid spot, the region withits label is extracted from the binary object mask to create thesingle-spot binary mask. The single-spot binary mask is applied to theoriginal object image to get the grayscale spot image of the respectivespot. The intensities of the pixels in the grayscale spot image aresummed to get the aggregate intensity of the spot 111. Once all thespots have been processed, the sum of all of the areas of the validspots are summed to get the aggregate spot area for the field 112. Theaggregate intensity of the spots is totaled to get the aggregate spotintensity of the field. There are several statistics to choose from forthe final score for the field (or well): (a) the number of valid spots;(b) the aggregate area of the valid spots; (c) the aggregate intensityof the valid spots; (d) the aggregate intensity of the valid spotsdivided by the total area of the nuclei. When more than one field isanalyzed within each well, the values for all the fields of the well areaveraged together to get an aggregate statistic for the well 113. (FIG.26)

The following examples of determining receptor internalization using theabove techniques illustrate the differences found between treated anduntreated cells. The nuclei of unstimulated cells are labeled with theDNA-specific Hoechst stain and imaged with a near-UV fluorescence filterset. The same cells are imaged with a blue fluorescence filter set whichshows the distribution of the GFP fluorescence. The nuclear mask isderived by applying a threshold to the nucleus-labeled image, and thebackground image is derived by the grayscale erosion and dilation of theGFP image, showing the variations in the background intensity. Theobject image is then derived by subtracting the background image fromthe GFP image, resulting in faint spots. The object mask is then derivedby applying the threshold to the object image. Some faint spots areeliminated by the thresholding. Some others have fewer pixels over thethreshold than the requirement for a valid spot. As a result, very fewvalid spots are found in the image of unstimulated cells. The spotcount, aggregate spot areas, and aggregate spot brightness all have lowvalues.

In a second example, the nuclei of stimulated cells are labeled with theDNA-specific Hoechst stain and imaged as in the preceding example. Thenuclear mask is derived by the automated thresholding method, and thebackground image is derived by the grayscale erosion and dilation of theGFP image, showing the variations in the background intensity. Theobject image is derived by subtracting the background image from the GFPimage, resulting in bright spots. The object mask is derived by applyingthe threshold to the object image. Many spots are seen in the objectmask, and many of those spots have enough pixels over the threshold tomeet the requirement for valid spots. The spot count, aggregate spotareas, and aggregate spot brightness all have high values. Results fromexperiments like these examples were shown previously in FIG. 25.

FIG. 29 shows a representative display of a PC screen showing data whichwas obtained by the methods described in the above examples. Each datapoint represents the Spot Count of a single well of the plate,calculated by summing together the Spot Counts of the fields of thewell. The graph 300 shows individual curves, each representing a singlerow of the 96 well plate. The leftmost six points of each curverepresent the Spot Counts of untreated wells, while the rightmost sixpoints represent treated wells. The Spot Count feature (“obj count” inillustration) can be selected using the list 302. The numerical valuesfor all the rows are shown in spreadsheet format 303. The graph 300 andspreadsheet 303 can be printed, and the spreadsheet can be exported in acomma-separated format for input into a spreadsheet program such asMicrosoft Excel™.

Alternatively, the data can be displayed on a field by field basis (FIG.30). Each graph at the top 304, 305, and 306 can be set to plot any oneof the computed statistics (averaged over the fields of the well) vs.the well number. The spreadsheet 307 shows the numerical data computedon a field by field basis. Selection of a line from the spreadsheetcauses display of the corresponding Hoechst 308 and GFP 309 images to bedisplayed. The spreadsheet 305 can be printed or exported in an ASCIIfile format for input into a spreadsheet program such as MicrosoftExcel™.

The graph 304 shows the Spot Count vs. the well number. The Spot Countis the number of valid spots detected in the input GFP images. Theinvention provides a computer means for converting the digital signalfrom the camera into this parameter and for plotting the parameter vs.the well number.

The graph 305 shows the aggregate spot area (“total spot area” inillustration) vs. the well number. The aggregate spot area is the summedareas of all valid spots detected in the input GFP images. The inventionprovides a computer means for converting the digital signal from thecamera into this parameter and for plotting this parameter vs. the wellnumber.

The graph 306 shows the normalized spot intensity ratio (“Spot IntenRatio×100” in illustration) vs. the well number. The normalized spotintensity ratio is the summed intensities of all the pixels in the validspots detected in the input GFP images, divided by the summed number ofpixels in the nucleus masks in the corresponding Hoechst image. Theinvention provides a computer means for converting the digital signalfrom the camera into this parameter and for plotting the parameter vs.the well number.

FIG. 25 is a graphical representation of data from validation runs ofthe PTHR internalization screen. The figure illustrates that the datafor min. (“minimum response”=unstimulated) and max. (“maximumresponse”=stimulated) are consistent between different plates (thedifferences are not statistically significant), giving c.o.v.'s(coefficients of variance) within a consistent and acceptable range.

In a specific example of a high-content screen, four fields wereacquired in each well. The Spot Count was summed across the fields of awell, and averaged among the similarly treated wells. The untreated halfof the plate had a Spot Count of 69.3±17.7 (mean±Standard Deviation)times the untreated half of the plate, giving a Coefficient of Variation(COV, the Standard Deviation divided by the mean) of 26%. The valuesfrom the fields of the treated half of the plate had a Spot Count of404.2±41.2, giving a COV of (10%). The mean Spot Count of the treatedhalf was 5.83 times the mean Spot Count of the untreated half.

EXAMPLE 5 Kinetic High Content Screen

Simply detecting the endpoint as internalized or not, may not besufficient for defining the potency of a compound as a receptor agonistor antagonist. In another embodiment, the cells are treated with drugand data are collected at various timepoints following drug treatment inorder to quantitate the kinetics of receptor internalization. Thesekinetic assays can be done on live cells as described above, ordifferent wells of cells can be fixed at each of the various timepointsof interest following drug treatment. In either case, cells can belabeled using the reagents and methods described above. Such kineticmeasurements would provide information not only about potency during thetime course of measurement, but would also allow extrapolation of thedata to much longer time periods.

In a preferred embodiment, kinetic measurements are first made in onechannel of fluorescence in a high-throughput or ultra-high-throughputmode for a cellular response associated with receptor internalization.This response is less receptor specific than the internalization processitself and may include, but is not limited to, changes in Ca²⁺, cAMP, orIP₃ concentrations, or activation of any of a variety of kinases. Wellsexhibiting the desired output from this parameter are then analyzed inthe HCS mode for highly detailed temporal and spatial information on acell-by-cell basis.

The luminescence signals of live or fixed cells are analyzed using acell scanning system, such as the cell scanning system of the presentinvention.

EXAMPLE 6 Inserted Sequences and their Ligands for High-Content ScreensIncorporating Dual-labeled Receptors

In another embodiment, a membrane receptor is modified to containspecific peptide sequences fused to each end in order to distinctlylabel the extracellular and intracellular domains. A ratio offluorescence intensity of the two labels is made in unstimulated andstimulated cells; since the amino terminus of the receptor is onlyavailable for labeling while the receptor is inserted in the plasmamembrane, the ratio of the two labels in unpermeabilized cells can beused to measure the extent of internalization of the receptor. There iscurrently no known technology for simultaneously measuring the relativeextracellular availability of external and internal domains of membranereceptors.

Appropriate cells are transiently or stably transfected with a DNAconstruct (either plasmid or viral based) that expresses the GPCR ofinterest fused to an epitope tag at its amino terminus and a molecularbased chromophore at its carboxy terminus. Alternatively, the GPCR maybe fused to an epitope tag at its carboxyl terminus and a molecularbased chromophore at its amino terminus. The expression of the GPCRfusion may be constitutive or inducible.

Some examples of epitope tags include, but are not limited to, FLAG(Sigma Chemical, St. Louis, Mo.), myc (9E10) (Invitrogen, Carlsbad,Calif.), 6-His (Invitrogen; Novagen, Madison, Wis.), and HA (BoehringerManheim Biochemicals). The expression the GPCR fusion may beconstitutive or inducible.

Examples of useful molecular-based chromophores include, but are notlimited to, GFP and any of its various mutants (Heim and Tsien (1996)Current Biology 6: 178-182; Zhang et al. (1996) Biochem. Biophys. Res.Comm. 227: 707-711). In addition, any of the luciferases and theirmutants could also be used. The use of a luciferase as part of achimeric target protein comprises a novel labeling technique since theexamples of use of this molecular-based chromophore to date haveincluded use as a reporter of gene activity (Yang et al. (1998) J. Biol.Chem. 273(17): 10763-10770; Peng et al. (1998) J. Biol. Chem. 273(27):17286-17295; Baldari et al. (1998) Biologicals 26(1): 1-5)) andconstruction of biosensors (Campbell and Patel (1983) Biochem. J. 216:185-194; Sala-Newby and Campbell (1992) FEBS Lett. 307: 241-244; Jenkinset al. (1990) Biochem. Soc. Trans. 18: 463-464) but not as a chimera formarking a particular protein target. Expression of the membraneprotein-luminescent protein fusion may be constitutive (driven by any ofa variety of promoters, including but not limited to, CMV, SV40, RSV,actin, EF) or inducible (driven by any of a number of induciblepromoters including, but not limited to, tetracycline, ecdysone,steroid-responsive).

Alternatively, cells are transiently or stably transfected with a DNAconstruct (either plasmid or viral based) that expresses the membraneprotein of interest fused to two distinct epitope tags, with one beingfused to each end of the membrane protein.

The external availability of the inserted sequences depends on theinternalization state of the receptor. That is, the ratio of theexternal availability of the inserted sequences provides a directmeasure of the magnitude of receptor internalization. This is ahigh-content screen incorporating dual-labeled receptors. The externalavailability of the inserted sequences can be measured using a singleapproach or a combination of several approaches:

1. One or more of the inserted sequences can be epitopes for specificantibodies. Antibody binding to the epitope can be measured usinghistochemical, radioactive, or fluorescence methods. Possible epitopesinclude, but are not limited to, those shown in Table I.

TABLE I PEPTIDE EPITOPES AND THEIR CORRESPONDING ANTIBODIES SEQUENCE IDANTIBODY EPITOPE NUMBER SOURCE FLAG MDYKDDDDK SEQ ID NO:1 Sigma MycEQKLISEEDL SEQ ID NO:2 Invitrogen, Boehringer- Mannheim Biochemical(BMB) 6-His HHHHHH SEQ ID NO:3 Invitrogen, BMB, Berkley Antibody Company(BAbCO) AU1 DTYRYI SEQ ID NO:4 BAbCO AU5 TDFYLK SEQ ID NO:5 BAbCO Glu—EEEEYMPME SEQ ID NO:6 BAbCO Glu HA YPYDVPDYA SEQ ID NO:7 BMB, BAbCO IRSNPDSEIARYIRS SEQ ID NO:8 BAbCO KT-3 KPPTPPPEPET SEQ ID NO:9 BAbCOProtein EDQVDPRLIDGK SEQ ID NO:10 BMB C VSV-G YTDIEMNRLGK SEQ ID NO:11BMB HSV QPELAPEDPED SEQ ID NO:12 Novagen T7 MASMTGGGQQMG SEQ ID NO:13Novagen V5 GKPIPNPLLGLDST SEQ ID NO:14 Invitrogen Xpress ™ DLYDDDDK SEQID NO:15 Invitrogen

2. The inserted sequences can code for fluorescent proteins. Besides thenatural fluorophores of trp, tyr, and phe that exist in many proteins,other fluorescent protein sequences can be inserted. The GFP sequence orone of its mutant variants can be inserted into the sequence coding forthe receptor. Sequences coding for luciferase and its mutant variantscan also be inserted. Any peptide sequence that codes for or interactswith a fluorophore can be used in this method. The inserted sequencescan be structured to express fluorescent proteins with differentfluorescent properties such that fluorescent signals from each can bemeasured independently.

3. The inserted sequences can code for peptides that bind small (<1000M_(r)) ligands with high affinity (K_(d)<10⁻⁹) and specificity. Thesesmall molecules then form a tight bridge to other molecules ormacromolecules that can be luminescently or radioactively labeled. Theinserted sequences can be structured to bind different bridgingmolecules that bind distinctly labeled molecules or macromolecules suchthat signals from each can be measured independently. For example, thepeptide sequence -HHHHHH- will bind a metal ion (e.g., Ni²⁺, Cu²⁺, etc.)that will form a tight bridge with a polydentate acetic acid moiety(e.g., nitriloacetic acid). The acid moiety can be covalently linked tomolecules that are luminescent, radioactive, or otherwise lightabsorbing. These molecules can be luminescent dyes or macromoleculessuch as proteins that contain a luminescent or radioactive label. Otherexamples of inserted peptide sequences are such that they have a highaffinity for other small molecules that include steroid hormones,vitamins, and carbohydrates that form a tight bridge to other moleculesor macromolecules that can be luminescently or radioactively labeled.

EXAMPLE 7 A Generalized Dual-labeled Receptor InternalizationHigh-Content Screen

A modified G-protein coupled receptor (GPCR of known function or orphan)is transfected into human epithelial kidney cells (HEK 293) where itslocalization provides a measure of internalization from the plasmamembrane. The modified GPCR contains an epitope (for example, FLAG)label at the N-terminus (extracellular) and a GFP-molecule at theC-terminus (intracellular). To measure GPCR internalization after ligandtreatment, cells are fixed and labeled with Hoechst 33342 (a DNA-bindingfluorescent dye) and a distinct luminescently labeled antibody againstthe epitope tag. A cell screening system, such as the cell screeningsystem of the present invention, using ratio imaging, is used tocalculate the internalization of the GPCR due to the loss ofGPCR-epitope from the external side of the plasma membrane and anincrease in GFP-only-labeled receptor within the cell. This approach tomeasuring ligand-induced receptor internalization is independent of theinternalization mechanism so it is therefore applicable to a wide rangeof receptors of both known and unknown function.

EXAMPLE 8 High-content Screen of Human Glucocorticoid ReceptorTranslocation

One class of HCS involves the drug-induced dynamic redistribution ofintracellular constituents. The human glucocorticoid receptor (hGR), asingle “sensor” in the complex environmental response machinery of thecell, binds steroid molecules that have diffused into the cell. Theligand-receptor complex translocates to the nucleus wheretranscriptional activation occurs (Htun et al., Proc. Natl. Acad. Sci.93:4845, 1996).

In general, hormone receptors are excellent drug targets because theiractivity lies at the apex of key intracellular signaling pathways.Therefore, a high-content screen of hGR translocation has distinctadvantage over in vitro ligand-receptor binding assays. The availabilityof up to two more channels of fluorescence in the cell screening systemof the present invention permits the screen to contain two additionalparameters in parallel, such as other receptors, other distinct targetsor other cellular processes.

Plasmid construct. A eukaryotic expression plasmid containing a codingsequence for a green fluorescent protein—human glucocorticoid receptor(GFP-hGR) chimera was prepared using GFP mutants (Palm et al., Nat.Struct. Biol. 4:361 (1997). The construct was used to transfect a humancervical carcinoma cell line (HeLa).

Cell preparation and transfection. HeLa cells (ATCC CCL-2) weretrypsinized and plated using DMEM containing 5% charcoal/dextran-treatedfetal bovine serum (FBS) (HyClone) and 1% penicillin-streptomycin(C-DMEM) 12-24 hours prior to transfection and incubated at 37° C. and5% CO₂. Transfections were performed by calcium phosphateco-precipitation (Graham and Van der Eb, Virology 52:456, 1973; Sambrooket al., (1989). Molecular Cloning: A Laboratory Manual, Second ed. ColdSpring Harbor Laboratory Press, Cold Spring Harbor, 1989) or withLipofectamine (Life Technologies, Gaithersburg, Md.). For the calciumphosphate transfections, the medium was replaced, prior to transfection,with DMEM containing 5% charcoal/dextran-treated FBS. Cells wereincubated with the calcium phosphate-DNA precipitate for 4-5 hours at37° C. and 5% CO₂, washed 3-4 times with DMEM to remove the precipitate,followed by the addition of C-DMEM.

Lipofectamine transfections were performed in serum-free DMEM withoutantibiotics according to the manufacturer's instructions (LifeTechnologies, Gaithersburg, Md.). Following a 2-3 hour incubation withthe DNA-liposome complexes, the medium was removed and replaced withC-DMEM. All transfected cells in 96-well microtiter plates wereincubated at 33° C. and 5% CO₂ for 24-48 hours prior to drug treatment.Experiments were performed with the receptor expressed transiently inHeLa cells.

Dexamethiasone induction of GFP-hGR translocation. To obtainreceptor-ligand translocation kinetic data, nuclei of transfected cellswere first labeled with 5 μg/ml Hoechst 33342 (Molecular Probes) inC-DMEM for 20 minutes at 33° C. and 5% CO₂. Cells were washed once inHank's Balanced Salt Solution (HBSS) followed by the addition of 100 nMdexamethasone in HBSS with 1% charcoal/dextran-treated FBS. To obtainfixed time point dexamethasone titration data, transfected HeLa cellswere first washed with DMEM and then incubated at 33° C. and 5% CO₂ for1 h in the presence of 0-1000 nM dexamethasone in DMEM containing 1%charcoal/dextran-treated FBS. Cells were analyzed live or they wererinsed with HBSS, fixed for 15 min with 3.7% formaldehyde in HBSS,stained with Hoechst 33342, and washed before analysis. Theintracellular GFP-hGR fluorescence signal was not diminished by thisfixation procedure.

Image acquisition and analysis. Kinetic data were collected by acquiringfluorescence image pairs (GFP-hGR and Hoechst 33342-labeled nuclei) fromfields of living cells at 1 min intervals for 30 min after the additionof dexamethasone. Likewise, image pairs were obtained from each well ofthe fixed time point screening plates 1 h after the addition ofdexamethasone. In both cases, the image pairs obtained at each timepoint were used to define nuclear and cytoplasmic regions in each cell.Translocation of GFP-hGR was calculated by dividing the integratedfluorescence intensity of GFP-hGR in the nucleus by the integratedfluorescence intensity of the chimera in the cytoplasm or as anuclear-cytoplasmic difference of GFP fluorescence. In the fixed timepoint screen this translocation ratio was calculated from data obtainedfrom at least 200 cells at each concentration of dexamethasone tested.Drug-induced translocation of GFP-hGR from the cytoplasm to the nucleuswas therefore correlated with an increase in the translocation ratio.

Results. FIG. 20 schematically displays the drug-induced cytoplasm 253to nucleus 252 translocation of the human glucocorticoid receptor. Theupper pair of schematic diagrams depicts the localization of GFP-hGRwithin the cell before 250 (A) and after 251 (B) stimulation withdexamethasone. Under these experimental conditions, the drug induces alarge portion of the cytoplasmic GFP-hGR to translocate into thenucleus. This redistribution is quantified by determining the integratedintensities ratio of the cytoplasmic and nuclear fluorescence in treated255 and untreated 254 cells. The lower pair of fluorescence micrographsshow the dynamic redistribution of GFP-hGR in a single cell, before 254and after 255 treatment. The HCS is performed on wells containinghundreds to thousands of transfected cells and the translocation isquantified for each cell in the field exhibiting GFP fluorescence.Although the use of a stably transfected cell line would yield the mostconsistently labeled cells, the heterogeneous levels of GFP-hGRexpression induced by transient transfection did not interfere withanalysis by the cell screening system of the present invention.

To execute the screen, the cell screening system scans each well of theplate, images a population of cells in each, and analyzes cellsindividually. Here, two channels of fluorescence are used to define thecytoplasmic and nuclear distribution of the GFP-hGR within each cell.Depicted in FIG. 21 is the graphical user interface of the cellscreening system near the end of a GFP-hGR screen. The user interfacedepicts the parallel data collection and analysis capability of thesystem. The windows labeled “Nucleus” 261 and “GFP-hGR” 262 show thepair of fluorescence images being obtained and analyzed in a singlefield. The window labeled “Color Overlay” 260 is formed bypseudocoloring the above images and merging them so the user canimmediately identify cellular changes. Within the “Stored ObjectRegions” window 265, an image containing each analyzed cell and itsneighbors is presented as it is archived. Furthermore, as the HCS dataare being collected, they are analyzed, in this case for GFP-hGRtranslocation, and translated into an immediate “hit” response. The 96well plate depicted in the lower window of the screen 267 shows whichwells have met a set of user-defined screening criteria. For example, awhite-colored well 269 indicates that the drug-induced translocation hasexceeded a predetermined threshold value of 50%. On the other hand, ablack-colored well 270 indicates that the drug being tested induced lessthan 10% translocation. Gray-colored wells 268 indicate “hits” where thetranslocation value fell between 10% and 50%. Row “E” on the 96 wellplate being analyzed 266 shows a titration with a drug known to activateGFP-hGR translocation, dexamethasone. This example screen used only twofluorescence channels. Two additional channels (Channels 3 263 and 4264) are available for parallel analysis of other specific targets, cellprocesses, or cytotoxicity to create multiple parameter screens.

There is a link between the image database and the information databasethat is a powerful tool during the validation process of new screens. Atthe completion of a screen, he user has total access to image andcalculated data (FIG. 22). The comprehensive data analysis package ofthe cell screening system allows the user to examine HCS data atmultiple levels. Images 276 and detailed data in a spread sheet 279 forindividual cells can be viewed separately, or summary data can beplotted. For example, the calculated results of a single parameter foreach cell in a 96 well plate are shown in the panel labeled Graph 1 275.By selecting a single point in the graph, the user can display theentire data set for a particular cell that is recalled from an existingdatabase. Shown here are the image pair 276 and detailed fluorescenceand morphometric data from a single cell (Cell #118, gray line 277). Thelarge graphical insert 278 shows the results of dexamethasoneconcentration on the translocation of GFP-hGR. Each point is the averageof data from at least 200 cells. The calculated EC₅₀ for dexamethasonein this assay is 2 nM.

A powerful aspect of HCS with the cell screening system is thecapability of kinetic measurements using multicolor fluorescence andmorphometric parameters in living cells. Temporal and spatialmeasurements can be made on single cells within a population of cells ina field. FIG. 23 shows kinetic data for the dexamethasone-inducedtranslocation of GFP-hGR in several cells within a single field. HumanHeLa cells transfected with GFP-hGR were treated with 100 nMdexamethasone and the translocation of GFP-hGR was measured over time ina population of single cells. The graph shows the response oftransfected cells 285, 286, 287, and 288 and non-transfected cells 289.These data also illustrate the ability to analyze cells with differentexpression levels.

EXAMPLE 9 High-content Screen of Drug-induced Apoptosis

Apoptosis is a complex cellular program that involves myriad molecularevents and pathways. To understand the mechanisms of drug action on thisprocess, it is essential to measure as many of these events within cellsas possible with temporal and spatial resolution. Therefore, anapoptosis screen that requires little cell sample preparation yetprovides an automated readout of several apoptosis-related parameterswould be ideal. A cell-based assay designed for the cell screeningsystem has been used to simultaneously quantify several of themorphological, organellar, and macromolecular hallmarks ofpaclitaxel-induced apoptosis.

Cell preparation. The cells chosen for this study were mouse connectivetissue fibroblasts (L-929; ATCC CCL-1) and a highly invasiveglioblastoma cell line (SNB-19; ATCC CRL-2219) (Welch et al., In VitroCell. Dev. Biol. 31:610, 1995). The day before treatment with anapoptosis inducing drug, 3500 cells were placed into each well of a96-well plate and incubated overnight at 37° C. in a humidified 5% CO₂atmosphere. The following day, the culture medium was removed from eachwell and replaced with fresh medium containing various concentrations ofpaclitaxel (0-50 μM) from a 20 mM stock made in DMSO. The maximalconcentration of DMSO used in these experiments was 0.25%. The cellswere then incubated for 26 h as above. At the end of the paclitaxeltreatment period, each well received fresh medium containing 750 nMMITOTRACKER® Red (Molecular Probes; Eugene, Oreg.) and 3 μg/ml Hoechst33342 DNA-binding dye (Molecular Probes) and was incubated as above for20 min. Each well on the plate was then washed with HBSS and fixed with3.7% formaldehyde in HBSS for 15 min at room temperature. Theformaldehyde was washed out with HBSS and the cells were permeabilizedfor 90 s with 0.5% (v/v) Triton X-100, washed with HBSS, incubated with2 U ml⁻¹ BODIPY® FL phallacidin (Molecular Probes) for 30 min, andwashed with HBSS. The wells on the plate were then filled with 200 μlHBSS, sealed, and the plate stored at 4° C. if necessary. Thefluorescence signals from plates stored this way were stable for atleast two weeks after preparation. As in the nuclear translocationassay, fluorescence reagents can be designed to convert this assay intoa live cell high-content screen.

Image acquisition and analysis on the ArrayScan System. The fluorescenceintensity of intracellular MITOTRACKER® Red, Hoechst 33342, and BODIPY®FL phallacidin was measured with the cell screening system as describedsupra. Morphometric data from each pair of images obtained from eachwell was also obtained to detect each object in the image field (e.g.,cells and nuclei), and to calculate its size, shape, and integratedintensity.

Calculations and output. A total of 50-250 cells were measured per imagefield. For each field of cells, the following calculations wereperformed: (1) The average nuclear area (μm²) was calculated by dividingthe total nuclear area in a field by the number of nuclei detected. (2)The average nuclear perimeter (μm) was calculated by dividing the sum ofthe perimeters of all nuclei in a field by the number of nuclei detectedin that field. Highly convoluted apoptotic nuclei had the largestnuclear perimeter values. (3) The average nuclear brightness wascalculated by dividing the integrated intensity of the entire field ofnuclei by the number of nuclei in that field. An increase in nuclearbrightness was correlated with increased DNA content. (4) The averagecellular brightness was calculated by dividing the integrated intensityof an entire field of cells stained with MITOTRACKER® dye by the numberof nuclei in that field. Because the amount of MITOTRACKER® dye thataccumulates within the mitochondria is proportional to the mitochondrialpotential, an increase in the average cell brightness is consistent withan increase in mitochondrial potential. (5) The average cellularbrightness was also calculated by dividing the integrated intensity ofan entire field of cells w stained with BODIPY® FL phallacidin dye bythe number of nuclei in that field. Because the phallotoxins bind withhigh affinity to the polymerized form of actin, the amount of BODIPY® FLphallacidin dye that accumulates within the cell is proportional toactin polymerization state. An increase in the average cell brightnessis consistent with an increase in actin polymerization.

Results. FIG. 24 (top panels) shows the changes paclitaxel induced inthe nuclear morphology of L-929 cells. Increasing amounts of paclitaxelcaused nuclei to enlarge and fragment 293, a hallmark of apoptosis.Quantitative analysis of these and other images obtained by the cellscreening system is presented in the same figure. Each parametermeasured showed that the L-929 cells 296 were less sensitive to lowconcentrations of paclitaxel than were SNB-19 cells 297. At higherconcentrations though, the L-929 cells showed a response for eachparameter measured. The multiparameter approach of this assay is usefulin dissecting the mechanisms of drug action. For example, the area,brightness, and fragmentation of the nucleus 298 and actinpolymerization values 294 reached a maximum value when SNB-19 cells weretreated with 10 nM paclitaxel (FIG. 24; top and bottom graphs). However,mitochondrial potential 295 was minimal at the same concentration ofpaclitaxel (FIG. 24; middle graph). The fact that all the parametersmeasured approached control levels at increasing paclitaxelconcentrations (>10 nM) suggests that SNB-19 cells have low affinitydrug metabolic or clearance pathways that are compensatory atsufficiently high levels of the drug. Contrasting the drug sensitivityof SNB-19 cells 297, L-929 showed a different response to paclitaxel296. These fibroblastic cells showed a maximal response in manyparameters at 5 μM paclitaxel, a 500-fold higher dose than SNB-19 cells.Furthermore, the L-929 cells did not show a sharp decrease inmitochondrial potential 295 at any of the paclitaxel concentrationstested. This result is consistent with the presence of unique apoptosispathways between a normal and cancer cell line. Therefore, these resultsindicate that a relatively simple fluorescence labeling protocol can becoupled with the cell screening system of the present invention toproduce a high-content screen of key events involved in programmed celldeath.

EXAMPLE 10 Protease Induced Translocation of a Signaling EnzymeContaining a Disease-associated Sequence from Cytoplasm to Nucleus

Plasmid construct. A eukaryotic expression plasmid containing a codingsequence for a green fluorescent protein—caspase (Cohen (1997),Biochemical J. 326:1-16; Liang et al. (1997), J. of Molec. Biol.274:291-302) chimera is prepared using GFP mutants. The construct isused to transfect eukaryotic cells.

Cell preparation and transfection. Cells are trypsinized and plated 24 hprior to transfection and incubated at 37° C. and 5% CO₂. Transfectionsare performed by methods including, but not limited to calcium phosphatecoprecipitation or lipofection. Cells are incubated with the calciumphosphate-DNA precipitate for 4-5 hours at 37° C. and 5% CO₂, washed 3-4times with DMEM to remove the precipitate, followed by the addition ofC-DMEM. Lipofectamine transfections are performed in serum-free DMEMwithout antibiotics according to the manufacturer's instructions.Following a 2-3 hour incubation with the DNA-liposome complexes, themedium is removed and replaced with C-DMEM.

Apopototic induction of Caspase-GFP translocation. To obtain Caspase-GFPtranslocation kinetic data, nuclei of transfected cells are firstlabeled with 5 μg/ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20minutes at 37° C. and 5% CO₂. Cells are washed once in Hank's BalancedSalt Solution (HBSS) followed by the addition of compounds that induceapoptosis. These compounds include, but are not limited to paclitaxel,staurosporine, ceramide, and tumor necrosis factor. To obtain fixed timepoint titration data, transfected cells are first washed with DMEM andthen incubated at 37° C. and 5% CO₂ for 1 h in the presence of 0-1000 nMcompound in DMEM. Cells are analyzed live or they are rinsed with HBSS,fixed for 15 min with 3.7% formaldehyde in HBSS, stained with Hoechst33342, and washed before analysis.

Image acquisition and analysis. Kinetic data are collected by acquiringfluorescence image pairs (Caspase-GFP and Hoechst 33342-labeled nuclei)from fields of living cells at 1 min intervals for 30 min after theaddition of compound. Likewise, image pairs are obtained from each wellof the fixed time point screening plates 1 h after the addition ofcompound. In both cases, the image pairs obtained at each time point areused to define nuclear and cytoplasmic regions in each cell.Translocation of Caspase-GFP is calculated by dividing the integratedfluorescence intensity of Caspase-GFP in the nucleus by the integratedfluorescence intensity of the chimera in the cytoplasm or as anuclear-cytoplasmic difference of GFP fluorescence. In the fixed timepoint screen this translocation ratio is calculated from data obtainedfrom at least 200 cells at each concentration of compound tested.Drug-induced translocation of Caspase-GFP from the cytoplasm to thenucleus is therefore correlated with an increase in the translocationratio. Molecular interaction libraries including, but not limited tothose comprising putative activators or inhibitors ofapoptosis-activated enzymes are use to screen the indicator cell linesand identify a specific ligand for the DAS, and a pathway activated bycompound activity.

EXAMPLE 11 Identification of Novel Steroid Receptors from DAS

Two sources of material and/or information are required to make use ofthis embodiment, which allows assessment of the function of anuncharacterized gene. First, disease associated sequence bank(s)containing cDNA sequences suitable for transfection into mammalian cellscan be used. Because every RADE or differential expression experimentgenerates up to several hundred sequences, it is possible to generate anample supply of DAS. Second, information from primary sequence databasesearches can be used to place DAS into broad categories, including, butnot limited to, those that contain signal sequences, seventrans-membrane motifs, conserved protease active site domains, or otheridentifiable motifs. Based on the information acquired from thesesources, algorithm types and indicator cell lines to be transfected areselected. A large number of motifs are already well characterized andencoded in the linear sequences contained within the large number genesin existing genomic databases.

In one embodiment, the following steps are taken:

1) Information from the DAS identification experiment (includingdatabase searches) is used as the basis for selecting the relevantbiological processes. (for example, look at the DAS from a tumor linefor cell cycle modulation, apoptosis, metastatic proteases, etc.)

2) Sorting of DNA sequences or DAS by identifiable motifs (ie. signalsequences, 7-transmembrane domains, conserved protease active sitedomains, etc.) This initial grouping will determine fluorescent taggingstrategies, host cell lines, indicator cell lines, and banks ofbioactive molecules to be screened, as described supra.

3) Using well established molecular biology methods, ligate DAS into anexpression vector designed for this purpose. Generalized expressionvectors contain promoters, enhancers, and terminators for which todeliver target sequences to the cell for transient expression. Suchvectors may also contain antibody tagging sequences, direct associationsequences, chromophore fusion sequences like GFP, etc. to facilitatedetection when expressed by the host.

4) Transiently transfect cells with DAS containing vectors usingstandard transfection protocols including: calcium phosphateco-precipitation, liposome mediated, DEAE dextran mediated, polycationicmediated, viral mediated, or electroporation, and plate into microtiterplates or microwell arrays. Alternatively, transfection can be donedirectly in the microtiter plate itself.

5) Carry out the cell screening methods as described supra.

In this embodiment, DAS shown to possess a motif(s) suggestive oftranscriptional activation potential (for example, DNA binding domain,amino terminal modulating domain, hinge region, or carboxy terminalligand binding domain) are utilized to identify novel steroid receptors.

Defining the fluorescent tags for this experiment involvesidentification of the nucleus through staining, and tagging the DAS bycreating a GFP chimera via insertion of DAS into an expression vector,proximally fused to the gene encoding GFP. Alternatively, a single chainantibody fragment with high affinity to some portion of the expressedDAS could be constructed using technology available in the art(Cambridge Antibody Technologies) and linked to a fluorophore (FITC) totag the putative transcriptional activator/receptor in the cells. Thisalternative would provide an external tag requiring no DNA transfectionand therefore would be useful if distribution data were to be gatheredfrom the original primary cultures used to generate the DAS.

Plasmid construct. A eukaryotic expression plasmid containing a codingsequence for a green fluorescent protein—DAS chimera is prepared usingGFP mutants. The construct is used to transfect HeLa cells. The plasmid,when transfected into the host cell, produces a GFP fused to the DASprotein product, designated GFP-DASpp.

Cell preparation and transfection. HeLa cells are trypsinized and platedusing DMEM containing 5% charcoal/dextran-treated fetal bovine serum(FBS) (Hyclone) and 1% penicillin-streptomycin (C-DMEM) 12-24 hoursprior to transfection and incubated at 37° C. and 5% CO₂. Transfectionsare performed by calcium phosphate coprecipitation or with Lipofectamine(Life Technologies). For the calcium phosphate transfections, the mediumis replaced, prior to transfection, with DMEM containing 5%charcoal/dextran-treated FBS. Cells are incubated with the calciumphosphate-DNA precipitate for 4-5 hours at 37° C. and 5% CO₂, and washed3-4 times with DMEM to remove the precipitate, followed by the additionof C-DMEM. Lipofectamine transfections are performed in serum-free DMEMwithout antibiotics according to the manufacturer's instructions.Following a 2-3 hour incubation with the DNA-liposome complexes, themedium is removed and replaced with C-DMEM. All transfected cells in96-well microtiter plates are incubated at 33° C. and 5% CO₂ for 24-48hours prior to drug treatment. Experiments are performed with thereceptor expressed transiently in HeLa cells.

Localization of expressed GFP-DASpp inside cells. To obtain cellulardistribution data, nuclei of transfected cells are first labeled with 5μg/ml Hoechst 33342 (Molecular Probes) in C-DMEM for 20 minutes at 33°C. and 5% CO₂. Cells are washed once in Hank's Balanced Salt Solution(HBSS). The cells are analyzed live or they are rinsed with HBSS, fixedfor 15 min with 3.7% formaldehyde in HBSS, stained with Hoechst 33342,and washed before analysis.

In a preferred embodiment, image acquisition and analysis are performedusing the cell screening system of the present invention. Theintracellular GFP-DASpp fluorescence signal is collected by acquiringfluorescence image pairs (GFP-DASpp and Hoechst 33342-labeled nuclei)from field cells. The image pairs obtained at each time point are usedto define nuclear and cytoplasmic regions in each cell. Datademonstrating dispersed signal in the cytoplasm would be consistent withknown steroid receptors that are DNA transcriptional activators.

Screening for induction of GFP-DASpp translocation. Using the aboveconstruct, confirmed for appropriate expression of the GFP-DASpp, as anindicator cell line, a screen of various ligands is performed using aseries of steroid type ligands including, but not limited to: estrogen,progesterone, retinoids, growth factors, androgens, and many othersteroid and steroid based molecules. Image acquisition and analysis areperformed using the cell screening system of the invention. Theintracellular GFP-DASpp fluorescence signal is collected by acquiringfluorescence image pairs (GFP-DASpp and Hoechst 33342-labeled nuclei)from fields cells. The image pairs obtained at each time point are usedto define nuclear and cytoplasmic regions in each cell. Translocation ofGFP-DASpp is calculated by dividing the integrated fluorescenceintensity of GFP-DASpp in the nucleus by the integrated fluorescenceintensity of the chimera in the cytoplasm or as a nuclear-cytoplasmicdifference of GFP fluorescence. A translocation from the cytoplasm intothe nucleus indicates a ligand binding activation of the DASpp thusidentifying the potential receptor class and action. Combining this datawith other data obtained in a similar fashion using known inhibitors andmodifiers of steroid receptors, would either validate the DASpp as atarget, or more data would be generated from various sources.

EXAMPLE 12 Additional Screens

Translocation Between the Plasma Membrane and the Cytoplasm:

Profilactin complex dissociation and binding of profilin to the plasmamembrane. In one embodiment, a fluorescent protein biosensor of profilinmembrane binding is prepared by labeling purified profilin (Federov etal.(1994), J. Molec. Biol. 241:480-482; Lanbrechts et al. (1995), Eur. JBiochem. 230:281-286) with a probe possessing a fluorescence lifetime inthe range of 2-300 ns. The labeled profilin is introduced into livingindicator cells using bulk loading methodology and the indicator cellsare treated with test compounds. Fluorescence anisotropy imagingmicroscopy (Gough and Taylor (1993), J. Cell Biol. 121:1095-1107) isused to measure test-compound dependent movement of the fluorescentderivative of profilin between the cytoplasm and membrane for a periodof time after treatment ranging from 0.1 s to 10 h.

Rho-RhoGDI complex translocation to the membrane. In another embodiment,indicator cells are treated with test compounds and then fixed, washed,and permeabilized. The indicator cell plasma membrane, cytoplasm, andnucleus are all labeled with distinctly colored markers followed byimmunolocalization of kilo protein (Self et al. (1995), Methods inEnzymology 256:3-10; Tanaka et al. (1995), Methods in Enzymology256:41-49) with antibodies labeled with a fourth color. Each of the fourlabels is imaged separately using the cell screening system, and theimages used to calculate the amount of inhibition or activation oftranslocation effected by the test compound. To do this calculation, theimages of the probes used to mark the plasma membrane and cytoplasm areused to mask the image of the immunological probe marking the locationof intracellular Rho protein. The integrated brightness per unit areaunder each mask is used to form a translocation quotient by dividing theplasma membrane integrated brightness/area by the cytoplasmic integratedbrightness/area. By comparing the translocation quotient values fromcontrol and experimental wells, the percent translocation is calculatedfor each potential lead compound.

β-Arrestin Translocation to the Plasma Membrane Upon G-protein ReceptorActivation.

In another embodiment of a cytoplasm to membrane translocationhigh-content screen, the translocation of β-arrestin protein from thecytoplasm to the plasma membrane is measured in response to celltreatment. To measure the translocation, living indicator cellscontaining luminescent domain markers are treated with test compoundsand the movement of the β-arrestin marker is measured in time and spaceusing the cell screening system of the present invention. In a preferredembodiment, the indicator cells contain luminescent markers consistingof a green fluorescent protein β-arrestin (GFP-β-arrestin) proteinchimera (Barak et al. (1997), J. Biol. Chem. 272:27497-27500; Daaka etal. (1998), J. Biol. Chem. 273:685-688) that is expressed by theindicator cells through the use of transient or stable cell transfectionand other reporters used to mark cytoplasmic and membrane domains. Whenthe indicator cells are in the resting state, the domain markermolecules partition predominately in the plasma membrane or in thecytoplasm. In the high-content screen, these markers are used todelineate the cell cytoplasm and plasma membrane in distinct channels offluorescence. When the indicator cells are treated with a test compound,the dynamic redistribution of the GFP-β-arrestin is recorded as a seriesof images over a time scale ranging from 0.1 s to 10 h. In a preferredembodiment, the time scale is 1 h. Each image is analyzed by a methodthat quantifies the movement of the GFP-β-arrestin protein chimerabetween the plasma membrane and the cytoplasm. To do this calculation,the images of the probes used to mark the plasma membrane and cytoplasmare used to mask the image of the GFP-β-arrestin probe marking thelocation of intracellular GFP-β-arrestin protein. The integratedbrightness per unit area under each mask is used to form a translocationquotient by dividing the plasma membrane integrated brightness/area bythe cytoplasmic integrated brightness/area. By comparing thetranslocation quotient values from control and experimental wells, thepercent translocation is calculated for each potential lead compound.The output of the high-content screen relates quantitative datadescribing the magnitude of the translocation within a large number ofindividual cells that have been treated with test compounds of interest.

Translocation Between the Endoplasmic Reticulum and the Golgi:

In one embodiment of an endoplasmic reticulum to Golgi translocationhigh-content screen, the translocation of a VSVG protein from the ts045mutant strain of vesicular stomatitis virus (Ellenberg et al. (1997), J.Cell Biol. 138:1193-1206; Presley et al. (1997) Nature 389:81-85) fromthe endoplasmic reticulum to the Golgi domain is measured in response tocell treatment. To measure the translocation, indicator cells containingluminescent reporters are treated with test compounds and the movementof the reporters is measured in space and time using the cell screeningsystem of the present invention. The indicator cells contain luminescentreporters consisting of a GFP-VSVG protein chimera that is expressed bythe indicator cell through the use of transient or stable celltransfection and other domain markers used to measure the localizationof the endoplasmic reticulum and Golgi domains. When the indicator cellsare in their resting state at 40° C., the GFP-VSVG protein chimeramolecules are partitioned predominately in the endoplasmic reticulum. Inthis high-content screen, domain markers of distinct colors used todelineate the endoplasmic reticulum and the Golgi domains in distinctchannels of fluorescence. When the indicator cells are treated with atest compound and the temperature is simultaneously lowered to 32° C.,the dynamic redistribution of the GFP-VSVG protein chimera is recordedas a series of images over a time scale ranging from 0.1 s to 10 h. Eachimage is analyzed by a method that quantifies the movement of theGFP-VSVG protein chimera between the endoplasmic reticulum and the Golgidomains. To do this calculation, the images of the probes used to markthe endoplasmic reticulum and the Golgi domains are used to mask theimage of the GFP-VSVG probe marking the location of intracellularGFP-VSVG protein. The integrated brightness per unit area under eachmask is used to form a translocation quotient by dividing theendoplasmic reticulum integrated brightness/area by the Golgi integratedbrightness/area. By comparing the translocation quotient values fromcontrol and experimental wells, the percent translocation is calculatedfor each potential lead compound. The output of the high-content screenrelates quantitative data describing the magnitude of the translocationwithin a large number of individual cells that have been treated withtest compounds of interest at final concentrations ranging from 10⁻¹² Mto 10⁻³ M for a period ranging from 1 min to 10 h.

Induction and Inhibition of Organellar Function:

Intracellular microtubule stability. In one embodiment of an organellarfunction high-content screen, the assembly state of intracellularmicrotubules is measured in response to cell treatment. To measuremicrotubule assembly state, indicator cells containing luminescentreporters are treated with test compounds and the distribution of thereporters is measured in space and time using the cell screening systemof the present invention.

In a preferred embodiment, the reporter of intracellular microtubuleassembly is MAP 4 (Bulinski et al. (1997), J. Cell Science110:3055-3064), a ubiquitous microtubule associated protein that isknown to interact with microtubules in interphase and mitotic cells. Theindicator cells contain luminescent reporters consisting of a GFP-MAP 4chimera that is expressed by the indicator cells through the use oftransient or stable cell transfection and other reporters are used tomeasure the localization of the cytoplasmic and membrane components. AGFP-MAP 4 construct is prepared as follows: PCR amplification of nativeor mutant GFP molecules using primers to introduce restriction enzymesites is performed. The PCR product is ligated into the MAP 4 cDNAwithin a eukaryotic expression vector. Indicator cells are thentransfected with the expression vector to produce either transiently orstably transfected indicator cells.

Indicator cells are treated with test compounds at final concentrationsranging from 10⁻¹² M to 10⁻³ M for a period ranging from 1 min to 10 h.Growth medium containing labeling reagent to mark the nucleus and thecytoplasm are added. After incubation, the cells are washed with Hank'sbalanced salt solution (HBSS), fixed with 3.7% formaldehyde for 10 minat room temperature, and washed and stored in HBSS.

Image data are obtained from both fixed and living indicator cells. Toextract morphometric data from each of the images obtained the followingmethod of analysis is used:

1. Threshold each nucleus and cytoplasmic image to produce a mask thathas value=0 for each pixel outside a nucleus or cell boundary.

2. Overlay the mask on the original image, detect each object in thefield (i.e., nucleus or cell), and calculate its size, shape, andintegrated intensity.

3. Overlay the whole cell mask obtained above on the correspondingGFP-MAP 4 image and use an automated measurement of edge strengthroutine (Kolega et al. (1993). BioImaging 1:136-150) to calculate thetotal edge strength within each cell. To normalize for cell size, thetotal edge strength is divided by the cell area to give a “fibrousness”value. Large fibrousness values are associated with strong edge strengthvalues and are therefore maximal in cells containing distinctmicrotubule structures. Likewise, small fibrousness values areassociated with weak edge strength and are minimal in cells withdepolymerized microtubules. The physiological range of fibrousnessvalues is set by treating cells with either the microtubule stabilizingdrug paclitaxel (10 μM) or the microtubule depolymerizing drugnocodazole (10 μg/ml).

High-content Screens Involving the Functional Localization ofMacromolecules

Within this class of high-content screen, the functional localization ofmacromolecules in response to external stimuli is measured within livingcells.

Glycolytic enzyme activity regulation. In a preferred embodiment of acellular enzyme activity high-content screen, the activity of keyglycolytic regulatory enzymes are measured in treated cells. To measureenzyme activity, indicator cells containing luminescent labelingreagents are treated with test compounds and the activity of thereporters is measured in space and time using cell screening system ofthe present invention.

In one embodiment, the reporter of intracellular enzyme activity isfructose-6-phosphate, 2-kinase/fructose-2,6-bisphosphatase (PFK-2), aregulatory enzyme whose phosphorylation state indicates intracellularcarbohydrate anabolism or catabolism (Deprez et al. (1997) J. Biol.Chem. 272:17269-17275; Kealer et al. (1996) FEBS Letters 395:225-227;Lee et al. (1996), Biochemistry 35:6010-6019). The indicator cellscontain luminescent reporters consisting of a fluorescent proteinbiosensor of PFK-2 phosphorylation. The fluorescent protein biosensor isconstructed by introducing an environmentally sensitive fluorescent dyenear to the known phosphorylation site of the enzyme (Deprez et al.(1997), supra; Giuliano et al. (1995), supra). The dye can be of theketocyanine class (Kessler and Wolfbeis (1991), Spectrochimica Acta47A:187-192) or any class that contains a protein reactive moiety and afluorochrome whose excitation or emission spectrum is sensitive tosolution polarity. The fluorescent protein biosensor is introduced intothe indicator cells using bulk loading methodology.

Living indicator cells are treated with test compounds, at finalconcentrations ranging from 10⁻¹² M to 10⁻³ M for times ranging from 0.1s to 10 h. In a preferred embodiment, ratio image data are obtained fromliving treated indicator cells by collecting a spectral pair offluorescence images at each time point. To extract morphometric datafrom each time point, a ratio is made between each pair of images bynumerically dividing the two spectral images at each time point, pixelby pixel. Each pixel value is then used to calculate the fractionalphosphorylation of PFK-2. At small fractional values of phosphorylation,PFK-2 stimulates carbohydrate catabolism. At high fractional values ofphosphorylation, PFK-2 stimulates carbohydrate anabolism.

Protein kinase A activity And localization of subunits. In anotherembodiment of a high-content screen, both the domain localization andactivity of protein kinase A (PKA) within indicator cells are measuredin response to treatment with test compounds.

The indicator cells contain luminescent reporters including afluorescent protein biosensor of PKA activation. The fluorescent proteinbiosensor is constructed by introducing an environmentally sensitivefluorescent dye into the catalytic subunit of PKA near the site known tointeract with the regulatory subunit of PKA (Harootunian et al. (1993),Mol. Biol. of the Cell 4:993-1002; Johnson et al. (1996), Cell85:149-158; Giuliano et al. (1995), supra). The dye can be of theketocyanine class (Kessler, and Wolfbeis (1991), Spectrochimica Acta47A:187-192) or any class that contains a protein reactive moiety and afluorochrome whose excitation or emission spectrum is sensitive tosolution polarity. The fluorescent protein biosensor of PKA activationis introduced into the indicator cells using bulk loading methodology.

In one embodiment, living indicator cells are treated with testcompounds, at final concentrations ranging from 10⁻¹² M to 10⁻³ M fortimes ranging from 0.1 s to 10 h. In a preferred embodiment, ratio imagedata are obtained from living treated indicator cells. To extractbiosensor data from each time point, a ratio is made between each pairof images, and each pixel value is then used to calculate the fractionalactivation of PKA (e.g., separation of the catalytic and regulatorysubunits after cAMP binding). At high fractional values of activity,PFK-2 stimulates biochemical cascades within the living cell.

To measure the translocation of the catalytic subunit of PKA, indicatorcells containing luminescent reporters are treated with test compoundsand the movement of the reporters is measured in space and time usingthe cell screening system. The indicator cells contain luminescentreporters consisting of domain markers used to measure the localizationof the cytoplasmic and nuclear domains. When the indicator cells aretreated with a test compounds, the dynamic redistribution of a PKAfluorescent protein biosensor is recorded intracellularly as a series ofimages over a time scale ranging from 0.1 s to 10 h. Each image isanalyzed by a method that quantifies the movement of the PKA between thecytoplasmic and nuclear domains. To do this calculation, the images ofthe probes used to mark the cytoplasmic and nuclear domains are used tomask the image of the PKA fluorescent protein biosensor. The integratedbrightness per unit area under each mask is used to form a translocationquotient by dividing the cytoplasmic integrated brightness/area by thenuclear integrated brightness/area. By comparing the translocationquotient values from control and experimental wells, the percenttranslocation is calculated for each potential lead compound. The outputof the high-content screen relates quantitative data describing themagnitude of the translocation within a large number of individual cellsthat have been treated with test compound in the concentration range of10⁻¹² M to 10⁻³ M.

High-content Screens Involving the Induction or Inhibition of GeneExpression

RNA-based Fluorescent Biosensors

Cytoskeletal protein transcription and message localization. Regulationof the general classes of cell physiological responses includingcell-substrate adhesion, cell-cell adhesion, signal transduction,cell-cycle events, intermediary and signaling molecule metabolism, celllocomotion, cell-cell communication, and cell death can involve thealteration of gene expression. High-content screens can also be designedto measure this lass of physiological response.

In one embodiment, the reporter of intracellular gene expression is anoligonucleotide that can hybridize with the target mRNA and alter itsfluorescence signal. In a preferred embodiment, the oligonucleotide is amolecular beacon (Tyagi and Kramer (1996) Nat. Biotechnol. 14:303-308),a luminescence-based reagent whose fluorescence signal is dependent onintermolecular and intramolecular interactions. The fluorescentbiosensor is constructed by introducing a fluorescence energy transferpair of fluorescent dyes such that there is one at each end (5′ and 3′)of the reagent. The dyes can be of any class that contains a proteinreactive moiety and fluorochromes whose excitation and emission spectraoverlap sufficiently to provide fluorescence energy transfer between thedyes in the resting state, including, but not limited to, fluoresceinand rhodamine (Molecular Probes, Inc.). In a preferred embodiment, aportion of the message coding for β-actin (Kislauskis et al. (1994), J.Cell Biol. 127:441-451; McCann et al. (1997), Proc. Natl. Acad. Sci.94:5679-5684; Sutoh (1982), Biochemistry 21:3654-3661) is inserted intothe loop region of a hairpin-shaped oligonucleotide with the endstethered together due to intramolecular hybridization. At each end ofthe biosensor a fluorescence donor (fluorescein) and a fluorescenceacceptor (rhodamine) are covalently bound. In the tethered state, thefluorescence energy transfer is maximal and therefore indicative of anunhybridized molecule. When hybridized with the mRNA coding for β-actin,the tether is broken and energy transfer is lost. The completefluorescent biosensor is introduced into the indicator cells using bulkloading methodology.

In one embodiment, living indicator cells are treated with testcompounds, at final concentrations ranging from 10⁻¹² M to 10⁻³ M fortimes ranging from 0.1 s to 10 h. In a preferred embodiment, ratio imagedata are obtained from living treated indicator cells. To extractmorphometric data from each time point, a ratio is made between eachpair of images, and each pixel value is then used to calculate thefractional hybridization of the labeled nucleotide. At small fractionalvalues of hybridization little expression of β-actin is indicated. Athigh fractional values of hybridization, maximal expression of β-actinis indicated. Furthermore, the distribution of hybridized moleculeswithin the cytoplasm of the indicator cells is also a measure of thephysiological response of the indicator cells.

Cell Surface Binding of a Ligand

Labeled insulin binding to its cell surface receptor in living cells.Cells whose plasma membrane domain has been labeled with a labelingreagent of a particular color are incubated with a solution containinginsulin molecules (Lee et al. (1997), Biochemistry 36:2701-2708;Martinez-Zaguilan et al. (1996), Am. J. Physiol. 270:C1438-C1446) thatare labeled with a luminescent probe of a different color for anappropriate time under the appropriate conditions. After incubation,unbound insulin molecules are washed away, the cells fixed and thedistribution and concentration of the insulin on the plasma membrane ismeasured. To do this, the cell membrane image is used as a mask for theinsulin image. The integrated intensity from the masked insulin image iscompared to a set of images containing known amounts of labeled insulin.The amount of insulin bound to the cell is determined from the standardsand used in conjunction with the total concentration of insulinincubated with the cell to calculate a dissociation constant or insulinto its cell surface receptor.

Labeling of Cellular Compartments

Whole Cell Labeling

Whole cell labeling is accomplished by labeling cellular components suchthat dynamics of cell shape and motility of the cell can be measuredover time by analyzing fluorescence images of cells.

In one embodiment, small reactive fluorescent molecules are introducedinto living cells. These membrane-permeant molecules both diffusethrough and react with protein components in the plasma membrane. Dyemolecules react with intracellular molecules to both increase thefluorescence signal emitted from each molecule and to entrap thefluorescent dye within living cells. These molecules include reactivechloromethyl derivatives of aminocoumarins, hydroxycoumarins, eosindiacetate, fluorescein diacetate, some BODIPY® dye derivatives, andtetramethylrhodamine. The reactivity of these dyes toward macromoleculesincludes free primary amino groups and free sulfhydryl groups.

In another embodiment, the cell surface is labeled by allowing the cellto interact with fluorescently labeled antibodies or lectins (SigmaChemical Company, St. Louis, Mo.) that react specifically with moleculeson the cell surface. Cell surface protein chimeras expressed by the cellof interest that contain a green fluorescent protein, or mutant thereof,component can also be used to fluorescently label the entire cellsurface. Once the entire cell is labeled, images of the entire cell orcell array can become a parameter in high content screens, involving themeasurement of cell shape, motility, size, and growth and division.

Plasma Membrane Labeling

In one embodiment, labeling the whole plasma membrane employs some ofthe same methodology described above for labeling the entire cells.Luminescent molecules that label the entire cell surface act todelineate the plasma membrane.

In a second embodiment subdomains of the plasma membrane, theextracellular surface, the lipid bilayer, and the intracellular surfacecan be labeled separately and used as components of high contentscreens. In the first embodiment, the extracellular surface is labeledusing a brief treatment with a reactive fluorescent molecule such as thesuccinimidyl ester or iodoacetamde derivatives of fluorescent dyes suchas the fluoresceins, rhodamines, cyanines, and BODIPYS®.

In a third embodiment, the extracellular surface is labeled usingfluorescently labeled macromolecules with a high affinity for cellsurface molecules. These include fluorescently labeled lectins such asthe fluorescein, rhodamine, and cyanine derivatives of lectins derivedfrom jack bean (Con A), red kidney bean (erythroagglutinin PHA-E), orwheat germ.

In a fourth embodiment, fluorescently labeled antibodies with a highaffinity for cell surface components are used to label the extracellularregion of the plasma membrane. Extracellular regions of cell surfacereceptors and ion channels are examples of proteins that can be labeledwith antibodies.

In a fifth embodiment, the lipid bilayer of the plasma membrane islabeled with fluorescent molecules. These molecules include fluorescentdyes attached to long chain hydrophobic molecules that interact stronglywith the hydrophobic region in the center of the plasma membrane lipidbilayer. Examples of these dyes include the PKH series of dyes (U.S.Pat. Nos. 4,783,401, 4,762701, and 4,859,584; available commerciallyfrom Sigma Chemical Company, St. Louis, Mo.), fluorescent phospholipidssuch as nitrobenzoxadiazole glycerophosphoethanolamine andfluorescein-derivatized dihexadecanoylglycerophosphoetha-nolamine,fluorescent fatty acids such as5-butyl-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene-3-nonanoic acid and1-pyrenedecanoic acid (Molecular Probes, Inc.), fluorescent sterolsincluding cholesteryl4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoateand cholesteryl 1-pyrenehexanoate, and fluorescently labeled proteinsthat interact specifically with lipid bilayer components such as thefluorescein derivative of annexin V (Caltag Antibody Co, Burlingame,Calif.).

In another embodiment, the intracellular component of the plasmamembrane is labeled with fluorescent molecules. Examples of thesemolecules are the intracellular components of the trimeric G-proteinreceptor, adenylyl cyclase, and ionic transport proteins. Thesemolecules can be labeled as a result of tight binding to a fluorescentlylabeled specific antibody or by the incorporation of a fluorescentprotein chimera that is comprised of a membrane-associated protein andthe green fluorescent protein, and mutants thereof.

Endosome Fluorescence Labeling

In one embodiment, ligands that are transported into cells byreceptor-mediated endocytosis are used to trace the dynamics ofendosomal organelles. Examples of labeled ligands include BODIPY®FL-labeled low density lipoprotein complexes, tetramethylrhodaminetransferrin analogs, and fluorescently labeled epidermal growth factor(Molecular Probes, Inc.)

In a second embodiment, fluorescently labeled primary or secondaryantibodies (Sigma Chemical Co. St. Louis, Mo.; Molecular Probes, Inc.Eugene, Oreg.; Caltag Antibody Co.) that specifically label endosomalligands are used to mark the endosomal compartment in cells.

In a third embodiment, endosomes are fluorescently labeled in cellsexpressing protein chimeras formed by fusing a green fluorescentprotein, or mutants thereof, with a receptor whose internalizationlabels endosomes. Chimeras of the EGF, transferrin, and low densitylipoprotein receptors are examples of these molecules.

Lysosome Labeling

In one embodiment, membrane permeant lysosome-specific luminescentreagents are used to label the lysosomal compartment of living and fixedcells. These reagents include the luminescent molecules neutral red,N-(3-((2,4-dinitrophenyl)amino)propyl)-N-(3-aminopropyl)methylamine, andthe LYSOTRACKER® probes which report intralysosomal pH as well as thedynamic distribution of lysosomes (Molecular Probes, Inc.)

In a second embodiment, antibodies against lysosomal antigens (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tolabel lysosomal components that are localized in specific lysosomaldomains. Examples of these components are the degradative enzymesinvolved in cholesterol ester hydrolysis, membrane protein proteases,and nucleases as well as the ATP-driven lysosomal proton pump.

In a third embodiment, protein chimeras consisting of a lysosomalprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelthe lysosomal domain. Examples of these components are the degradativeenzymes involved in cholesterol ester hydrolysis, membrane proteinproteases, and nucleases as well as the ATP-driven lysosomal protonpump.

Cytoplasmic Fluorescence Labeling

In one embodiment, cell permeant fluorescent dyes (Molecular Probes,Inc.) with a reactive group are reacted with living cells. Reactive dyesincluding monobromobimane, 5-chloromethylfluorescein diacetate, carboxyfluorescein diacetate succinimidyl ester, and chloromethyltetramethylrhodamine are examples of cell permeant fluorescent dyes thatare used for long term labeling of the cytoplasm of cells.

In a second embodiment, polar tracer molecules such as Lucifer yellowand cascade blue-based fluorescent dyes (Molecular Probes, Inc.) areintroduced into cells using bulk loading methods and are also used forcytoplasmic labeling.

In a third embodiment, antibodies against cytoplasmic components (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tofluorescently label the cytoplasm. Examples of cytoplasmic antigens aremany of the enzymes involved in intermediary metabolism. Enolase,phosphofructokinase, and acetyl-CoA dehydrogenase are examples ofuniformly distributed cytoplasmic antigens.

In a fourth embodiment, protein chimeras consisting of a cytoplasmicprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelthe cytoplasm. Fluorescent chimeras of uniformly distributed proteinsare used to label the entire cytoplasmic domain. Examples of theseproteins are many of the proteins involved in intermediary metabolismand include enolase, lactate dehydrogenase, and hexokinase.

In a fifth embodiment, antibodies against cytoplasmic antigens (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tolabel cytoplasmic components that are localized in specific cytoplasmicsub-domains. Examples of these components are the cytoskeletal proteinsactin, tubulin, and cytokeratin. A population of these proteins withincells is assembled into discrete structures, which in this case, arefibrous. Fluorescence labeling of these proteins with antibody-basedreagents therefore labels a specific sub-domain of the cytoplasm.

In a sixth embodiment, non-antibody-based fluorescently labeledmolecules that interact strongly with cytoplasmic proteins are used tolabel specific cytoplasmic components. One example is a fluorescentanalog of the enzyme DNAse I (Molecular Probes, Inc.) Fluorescentanalogs of this enzyme bind tightly and specifically to cytoplasmicactin, thus labeling a sub-domain of the cytoplasm. In another example,fluorescent analogs of the mushroom toxin phalloidin or the drugpaclitaxel (Molecular Probes, Inc.) are used to label components of theactin- and microtubule-cytoskeletons, respectively.

In a seventh embodiment, protein chimeras consisting of a cytoplasmicprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelspecific domains of the cytoplasm. Fluorescent chimeras of highlylocalized proteins are used to label cytoplasmic sub-domains. Examplesof these proteins are many of the proteins involved in regulating thecytoskeleton. They include the structural proteins actin, tubulin, andcytokeratin as well as the regulatory proteins microtubule associatedprotein 4 and α-actinin.

Nuclear Labeling

In one embodiment, membrane permeant nucleic-acid-specific luminescentreagents (Molecular Probes, Inc.) are used to label the nucleus ofliving and fixed cells. These reagents include cyanine-based dyes (e.g.,TOTO®, YOYO®, and BOBO™), phenanthidines and acridines (e.g., ethidiumbromide, propidium iodide, and acridine orange), indoles and imidazoles(e.g., Hoechst 33258, Hoechst 33342, and 4′,6-diamidino-2-phenylindole),and other similar reagents (e.g., 7-aminoactinomycin D,hydroxystilbamidine, and the psoralens).

In a second embodiment, antibodies against nuclear antigens (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tolabel nuclear components that are localized in specific nuclear domains.Examples of these components are the macromolecules involved inmaintaining DNA structure and function. DNA, RNA, histones, DNApolymerase, RNA polymerase, lamins, and nuclear variants of cytoplasmicproteins such as actin are examples of nuclear antigens.

In a third embodiment, protein chimeras consisting of a nuclear proteingenetically fused to an intrinsically luminescent protein such as thegreen fluorescent protein, or mutants thereof, are used to label thenuclear domain. Examples of these proteins are many of the proteinsinvolved in maintaining DNA structure and function. Histones, DNApolymerase, RNA polymerase, lamins, and nuclear variants of cytoplasmicproteins such as actin are examples of nuclear proteins.

Mitochondrial Labeling

In one embodiment, membrane permeant mitochondrial-specific luminescentreagents (Molecular Probes, Inc.) are used to label the mitochondria ofliving and fixed cells. These reagents include rhodamine 123,tetramethyl rosamine, JC-1, and the MITOTRACKER® reactive dyes.

In a second embodiment, antibodies against mitochondrial antigens (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tolabel mitochondrial components that are localized in specificmitochondrial domains. Examples of these components are themacromolecules involved in maintaining mitochondrial DNA structure andfunction. DNA, RNA, histones, DNA, polymerase, RNA polymerase, andmitochondrial variants of cytoplasmic macromolecules such asmitochondrial tRNA and rRNA are examples mitochondrial antigens. Otherexamples of mitochondrial antigens are the components of the oxidativephosphorylation system found in the mitochondria (e.g., cytochrome c,cytochrome c oxidase, and succinate dehydrogenase).

In a third embodiment, protein chimeras consisting of a mitochondrialprotein genetically fused to an intrinsically luminescent protein suchas the green fluorescent protein, or mutants thereof, are used to labelthe mitochondrial domain. Examples of these components are themacromolecules involved in maintaining mitochondrial DNA structure andfunction. Examples include histones, DNA polymerase, RNA polymerase, andthe components of the oxidative phosphorylation system found in themitochondria (e.g., cytochrome c, cytochrome c oxidase, and succinatedehydrogenase).

Endoplasmic Reticulum Labeling

In one embodiment, membrane permeant endoplasmic reticulum-specificluminescent reagents (Molecular Probes, Inc.) are used to label theendoplasmic reticulum of living and fixed cells. These reagents includeshort chain carbocyanine dyes (e.g., DiOC₆ and DiOC₃), long chaincarbocyanine dyes (e.g., DiIC₁₆ and DiIC₁₈), and luminescently labeledlectins such as concanavalin A.

In a second embodiment, antibodies against endoplasmic reticulumantigens (Sigma Chemical Co.; Molecular Probes, Inc.; Caltag AntibodyCo.) are used to label endoplasmic reticulum components that arelocalized in specific endoplasmic reticulum domains. Examples of thesecomponents are the macromolecules involved in the fatty acid elongationsystems, glucose-6-phosphatase, and HMG CoA-reductase.

In a third embodiment, protein chimeras consisting of a endoplasmicreticulum protein genetically fused to an intrinsically luminescentprotein such as the green fluorescent protein, or mutants thereof, areused to label the endoplasmic reticulum domain. Examples of thesecomponents are the macromolecules involved in the fatty acid elongationsystems, glucose-6-phosphatase, and HMG CoA-reductase.

Golgi Labeling

In one embodiment, membrane permeant Golgi-specific luminescent reagents(Molecular Probes, Inc.) are used to label the Golgi of living and fixedcells. These reagents include luminescently labeled macromolecules suchas wheat germ agglutinin and Brefeldin A as well as luminescentlylabeled ceramide.

In a second embodiment, antibodies against Golgi antigens (SigmaChemical Co.; Molecular Probes, Inc.; Caltag Antibody Co.) are used tolabel Golgi components that are localized in specific Golgi domains.Examples of these components are N-acetylglucosamine phosphotransferase,Golgi-specific phosphodiesterase, and mannose-6-phosphate receptorprotein.

In a third embodiment, protein chimeras consisting of a Golgi proteingenetically fused to an intrinsically luminescent protein such as thegreen fluorescent protein, or mutants thereof, are used to label theGolgi domain. Examples of these components are N-acetylglucosaminephosphotransferase, Golgi-specific phosphodiesterase, andmannose-6-phosphate receptor protein.

While many of the examples presented involve the measurement of singlecellular processes, this is again is intended for purposes ofillustration only. Multiple, parameter high-content screens can beproduced by combining several single parameter screens into amultiparameter high-content screen or by adding cellular parameters toany existing high-content screen. Furthermore, while each example isdescribed as being based on either live or fixed cells, eachhigh-content screen can be designed to be used with both live and fixedcells.

Those skilled in the art will recognize a wide variety of distinctscreens that can be developed based on the disclosure provided herein.There is a large and growing list of known biochemical and molecularprocesses in cells that involve translocations or reorganizations ofspecific components within cells. The signaling pathway from the cellsurface to target sites within the cell involves the translocation ofplasma membrane-associated proteins to the cytoplasm. For example, it isknown that one of the src family of protein tyrosine kinases, pp60c-src(Walker et al (1993), J. Biol. Chem. 268:19552-19558) to translocatesfrom the plasma membrane to the cytoplasm upon stimulation offibroblasts with platelet-derived growth factor (PDGF). Additionally,the targets for screening can themselves be converted intofluorescence-based reagents that report molecular changes includingligand-binding and post-translocational modifications.

15 1 9 PRT Artificial Sequence epitope tag 1 Met Asp Tyr Lys Asp Asp AspAsp Lys 1 5 2 10 PRT Artificial Sequence EPITOPE TAG 2 Glu Gln Lys LeuIle Ser Glu Glu Asp Leu 1 5 10 3 6 PRT Artificial Sequence epitope tag 3His His His His His His 1 5 4 6 PRT Artificial Sequence epitope tag 4Asp Thr Tyr Arg Tyr Ile 1 5 5 6 PRT Artificial Sequence epitope tag 5Thr Asp Phe Tyr Leu Lys 1 5 6 9 PRT Artificial Sequence epitope tag 6Glu Glu Glu Glu Tyr Met Pro Met Glu 1 5 7 9 PRT Artificial Sequenceepitope tag 7 Tyr Pro Tyr Asp Val Pro Asp Tyr Ala 1 5 8 12 PRTArtificial Sequence epitope tag 8 Asn Pro Asp Ser Glu Ile Ala Arg TyrIle Arg Ser 1 5 10 9 11 PRT Artificial Sequence epitope tag 9 Lys ProPro Thr Pro Pro Pro Glu Pro Glu Thr 1 5 10 10 12 PRT Artificial Sequenceepitope tag 10 Glu Asp Gln Val Asp Pro Arg Leu Ile Asp Gly Lys 1 5 10 1111 PRT Artificial Sequence epitope tag 11 Tyr Thr Asp Ile Glu Met AsnArg Leu Gly Lys 1 5 10 12 11 PRT Artificial Sequence epitope tag 12 GlnPro Glu Leu Ala Pro Glu Asp Pro Glu Asp 1 5 10 13 12 PRT ArtificialSequence epitope tag 13 Met Ala Ser Met Thr Gly Gly Gly Gln Gln Met Gly1 5 10 14 14 PRT Artificial Sequence epitope tag 14 Gly Lys Pro Ile ProAsn Pro Leu Leu Gly Leu Asp Ser Thr 1 5 10 15 8 PRT Artificial Sequenceepitope tag 15 Asp Leu Tyr Asp Asp Asp Asp Lys 1 5

We claim:
 1. An automated method for identifying compounds that modulateinternalization of cell surface receptor proteins comprising: providingan array of locations containing multiple cells to be treated with atest compound, wherein the cells possess at least a first luminescentlylabeled reporter molecule that reports on a cell surface receptorprotein of interest and at least a second luminescently labeled reportermolecule that identifies cells, and wherein the cell surface receptorprotein is either expressed as a luminescently labeled protein, or isluminescently labeled by contacting the cell with a luminescentlylabeled molecule that binds to the cell surface receptor protein,wherein the contacting can be carried out either before or aftertreatment with the test compound; contacting the cells with the at leasta second luminescently labeled reporter molecule, wherein the contactingcan be carried out either before or after treatment with the testcompound; treating the cells with the test compound; imaging multiplecells in the array of locations; automatically obtaining luminescentsignals from the at least second luminescently labeled reporter moleculeto identify cells; automatically obtaining luminescent signals from theat least first luminescently-labeled reporter molecule on or in thecells, comprising: a) acquiring an object image of the at least firstluminescently labeled reporter molecule on or in the individual cells;b) by segmenting the object image to produce a segmented object image;and c) calculating whether objects in the segmented object image meet orsurpass a user-defined threshold luminescent intensity value, wherein anobject that meets or surpasses a user-defined threshold luminescentintensity value represents an internalized cell surface receptor proteinof interest: converting the luminescent signals into digital data; andautomatically calculating from the digital data: i) a total number ofcells, calculated from the digital data from the at least secondluminescently labeled reporter molecule; and ii) a number and/or percentof cells that internalized the at least first luminescently labeledreporter molecule; wherein the calculations provide a measure of whetherthe test compound has modulated internalization of the luminescentlylabeled cell surface receptor protein.
 2. The method of claim 1, furthercomprising: treating the cells with a ligand that causes the cellsurface receptor protein to be internalized in the absence of the testcompound; and determining whether the test compound has inhibitedligand-induced internalization or the at least first luminescentlylabeled reporter molecule into the cell.
 3. The method of claim 1wherein the calculating of the total cell number comprises the steps of:a) acquiring an image of the cell nuclei; b) segmenting the image of thecell nuclei; and c) either (1) calculating the total area of all thenuclei in the image of the cell nuclei, or (2) counting a number ofnuclei.
 4. The method of claim 1 or 2 wherein sub-regions of the arrayof locations containing multiple cells are sampled multiple times atintervals to provide kinetic measurement of the modulation of cellsurface receptor protein internalization into the cell.
 5. The method ofclaim 1 further comprising determining one or more of the following: A)an aggregate area of the objects that represent the internalized cellsurface receptor protein of interest; B) an aggregate intensity of theobjects that represent the internalized cell surface receptor protein ofinterest; C) a normalized aggregate intensity of the objects thatrepresent the internalized cell surface receptor protein of interest; D)a number of the objects that represent the internalized cell surfacereceptor protein of interest; and E) an average number per cell of theobjects that represent the internalized cell surface receptor protein ofinterest.
 6. The method of claim 1, wherein the imaging multiple cellsin the array of locations comprises: i) obtaining a low resolution imageto identify locations in the array of locations that containinternalized cell surface receptor proteins; and ii) obtaining a highresolution image of only those locations that contain internalized cellsurface receptor proteins.